Hereditary neutropenia: dogs explain human neutrophil elastase mutations

doi:10.1016/j.molmed.2004.02.002

Trends in Molecular Medicine

Volume 10, Issue 4, 1 April 2004, Pages 163-170

https://doi.org/10.1016/j.molmed.2004.02.002 Get rights and content

Abstract

Mutations in ELA2, the gene encoding neutrophil elastase (NE), cause the human diseases cyclic neutropenia (CN) and severe congenital neutropenia (SCN). Numerous mutations are known, but their lack of consistent biochemical effect has proven puzzling. The recent finding that mutation of AP3B1, which encodes the β subunit of adaptor protein complex 3 (AP3), is the cause of canine CN suggests a model for the molecular basis of hereditary neutropenias, involving the mistrafficking of NE: AP3 recognizes NE as a cargo protein, and their interaction implies that NE is a transmembrane protein. Computerized algorithms predict two NE transmembrane domains. Most CN mutations fall within predicted transmembrane domains and lead to excessive deposition of NE in granules, whereas SCN mutations usually disrupt the AP3 recognition sequence, resulting in excessive transport to the plasma membrane.

Section snippets

The biology of the ELA2 gene product, NE

ELA2 is transcribed only in pro-myelocytic and pro-monocytic progenitors in the bone marrow, but the protein, if not the transcript, persists in the cells through terminal differentiation to neutrophils and monocytes [21]. ELA2 encodes a 267-residue protein that is post-translationally processed at both termini. The N-terminus contains a 27-residue pre sequence cleaved by a signal peptidase. The protease cathepsin C, also known as dipeptidyl peptidase I (DPPI), cleaves a remaining two residue

AP3B1 mutations as the cause of canine CN

Canine CN [27] is also known as gray Collie syndrome, because it arose in Collies and the dogs have a diluted coat color. The human and canine forms of the disease differ in a number of ways: human CN lacks pigmentary abnormalities, canine disease demonstrates autosomal-recessive transmission, in dogs, neutrophil counts cycle every two weeks rather than three weeks (Figure 1) and all blood cells cycle.

Using direct DNA sequencing and genetic linkage studies, canine ELA2 mutations were excluded

Interaction between the gene products responsible for human and canine CN

Given the similarities between canine and human CN, an obvious question is whether NE interacts with AP3? Specifically, does NE bind to μ3a or β3a, the subunits responsible for recognizing cargo proteins? A yeast two-hybrid assay established for testing adaptor protein subunit and cargo protein interactions [39], indicates that μ3a interacts with NE via a tyrosine-based recognition signal upstream of the C-terminal tail, but only after the C-terminal tail is removed [26]. Because the C-terminus

NE mutations aligning with predicted transmembrane regions

Previously, NE was recognized as a soluble protein, and its crystal structure supports its behavior as a textbook serine protease [40]. However, would computerized algorithms identifying transmembrane domains detect their presence in human NE? Surprisingly, most programs predict two transmembrane domains [26], each of which is bracketed by disulfide bonds (Figure 2a). Strikingly, when the mutations responsible for hereditary neutropenia are superimposed on the predicted transmembrane domains, a

Pathogenic targets of NE

What are the substrates of mislocalized NE? Any answer to this question must ultimately account for the periodicity observed in CN. Many groups have proposed that oscillations suggest a feedback circuit in which mature neutrophils inhibit progenitor cells [60]. Inhibition of progenitors leads to the loss of successive cohorts of maturing cells, eventually depleting the generation of cells producing the inhibitory signal, thus allowing the pattern to repeat. It is, therefore, expected that the

Concluding remarks

NE has a significant role in myelopoiesis, and its previously underappreciated localization in cellular compartments other than granules, with attendant implications for NE as a membrane protein, opens a search for identifying important regulatory targets. This has far-reaching implications for serine protease biochemistry, subcellular trafficking, hematopoiesis and the pathogenesis of neutropenia and leukemia. Outstanding questions remain (Box 3) and future studies will aim to answer them.

Acknowledgements

We thank David Ehlert for assistance preparing figures, the estate of Dr Lee Ford for access to her notebooks, and Dr Richard Swank for mouse and Ginny Cuneo for dog photographs. Supported by grants (to M.H.) from the NIH (DK55820, DK58161) and Burroughs-Wellcome Fund (SATR-1002189).

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Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.
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Neutropenia is common to both Hermansky-Pudlak syndrome type 2 and canine cyclic hematopoiesis (CH) which are caused by mutations in the AP3B1 gene. The purpose of this study was to determine if pearl mice were neutropenic. Complete blood counts (CBCs) and bone marrow differential counts, colony forming unit (CFU) assay, bone marrow lineage negative (lin^–), Sca⁺ and c-kit⁺ cells (LSK cells), bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were compared in C57Bl6 (Bl/6) and pearl mice. Stress granulopoiesis was evaluated following 200 mg/kg cyclophosphamide or 1 mg/kg bortezomib administration and by limiting dilution bone marrow transplantation. The CBCs and CFUs were determined in Bl/6 and pearl mice following AMD3100 or granulocyte colony-stimulating factor (G-CSF) administration. Pearl mice were not neutropenic and did not have cyclic neutropenia. Bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were similar in pearl and Bl/6 mice. The numbers of CFU-G, CFU-GEMM, and LSK cells were increased moderately in pearl mice. Stress granulopoiesis was similar in Bl/6 and pearl mice. CFU assays and CBCs performed on Bl/6 and pearl mice administered AMD3100 resulted in similar results. However, normal mice administered G-CSF had higher peripheral blood neutrophil counts and greater CFU numbers compared with pearl mice. Unlike patients with HPS-2 and dogs with CH, pearl mice did not have neutropenia or CH but had decreased hematopoietic progenitor cell and granulocyte mobilization in response to G-CSF.
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Citation Excerpt :
However, the tyrosine-based signal in human ELA2, which interacts with AP-3 (43), is not conserved in canine ELA2 (GenBankTM accession number AF494190). This suggests that a different signal motif, such as a dileucine signal that is recognized by AP-3 (44), is used to target ELA2 to lysosomes in dogs. Moreover, clathrin-mediated endocytosis of bovine AE1 uses a noncanonical YXXXΦ motif instead of the YXXΦ signal (34), and this is another example of variability in the sequences of motifs used for cargo recognition.
Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), ²⁶VSIPM³⁰ in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues ⁸⁹⁵LIL⁸⁹⁷ (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to ⁸⁹⁵AAA⁸⁹⁷, specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C.
Endoplasmic reticulum (ER) export of transmembrane proteins depends on the interaction between cargo signals and Sec24 isoforms.
The ΦXΦXΦ sequence facilitates the ER export of model proteins and selectively binds to Sec24C.
ΦXΦXΦ is a novel ER export signal that is specifically recognized by Sec24C.
This novel cargo-Sec24 interaction provides mechanistic insights into vesicular transport.

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Trends in Molecular Medicine

Hereditary neutropenia: dogs explain human neutrophil elastase mutations

Abstract

Section snippets

The biology of the ELA2 gene product, NE

AP3B1 mutations as the cause of canine CN

Interaction between the gene products responsible for human and canine CN

NE mutations aligning with predicted transmembrane regions

Pathogenic targets of NE

Concluding remarks

Acknowledgements

Blood

Blood

Blood

Immunity

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell

Semin. Cell Dev. Biol.

Gene

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Blood

J. Biol. Chem.

Cell

Blood

Biochem. Biophys. Res. Commun.

Am. J. Hum. Genet.

Blood

Exp. Hematol.

Dev. Biol.

J. Mol. Biol.

Familial cyclical neutropenia

Br. J. Haematol.

Cyclic hematopoiesis: human cyclic neutropenia

Exp. Hematol.

Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis

Nat. Genet.

Leukemia in severe congenital neutropenia: defective proteolysis suggests new pathways to malignancy and opportunities for therapy

Cancer Invest.

Retraction of Aprikyan et al

Blood

Mutations of the granulocyte-colony stimulating factor receptor in patients with severe congenital neutropenia are not required for transformation to acute myeloid leukaemia and may be a bystander phenomenon

Br. J. Haematol.

The presence of an ELA2 mutation correlates with a more severe expression of neutropenia: study of 81 patients from the French severe chronic neutropenia register

Blood

Dysregulation of transcriptions in primary granule constituents during myeloid proliferation and differentiation in patients with severe congenital neutropenia

J. Leukoc. Biol.

Neutrophil elastase gene mutations in cyclic neutropenia

Turk. J. Pediatr.

Mutations in the gene encoding neutrophil elastase (ELA2) are not sufficient to cause the phenotype of congenital neutropenia

Br. J. Haematol.

Possibility of somatic mosaicism of ELA2 mutation overlooked in an asymptomatic father transmitting severe congenital neutropenia to two offspring

Br. J. Haematol.

Reply to Benson and Horwitz

Br. J. Haematol.

Gfi-1 oncoproteins in hematopoiesis

Hematology

Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1

Nat. Genet.