JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia

Abstract

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.

Figure 1

Identification of mutations in JAGN1. (a) Genotypes of affected (filled symbols) and unaffected individuals (open symbols) from Family A on chromosome 3p. (b) Exon/intron scheme of the JAGN1 gene locus with all mutations identified in this study. (c) JAGN1 protein expression was determined by Western blot analysis using an antibody directed against the N-terminal end of JAGN1 as outlined in Material and Methods. Px denotes patient number x in this study, PxF the respective father.

Figure 2

Phenotype of JAGN1-mutant neutrophils. (a) Transmission electron microscopy (TEM) of the bone marrow. Myelocytes (left panels) from patients showed aberrant, enlarged endoplasmic reticulum structures (arrows in left panels) in contrast to myelocytes from a healthy donor (HD). Differentiated granulocytes from patients (right panels) showed a paucity of typical granules (arrows in the right panel). (b) Increased protein expression of BIP in JAGN1-mutated granulocytes from patients P7 and P8. (c) MALDI-TOF MS partial spectra of permethylated N-glycans isolated from a heterozygous carrier (healthy control; mother) and patient P8 with JAGN1 mutation. The structures detected at m/z 3,402, 3,851, 4,212, 4,300 and 4,749 were found in reduced abundance in the patients compared to healthy controls, while those detected at m/z 4,025, 4,474 and 4,924 in healthy controls were almost undetectable in the patients. Red peaks depict the glycans whose abundance is markedly altered in the patient. For clarity, black peaks in the patient’s spectrum are not annotated. See the upper panel for annotations. Structure assignments are based on composition, tandem MS, and biosynthetic knowledge. (d) Increased apoptosis in JAGN1-mutant neutrophils (P12) in response to staurosporine as determined by flow cytometric analysis using Annexin-V and propidium iodide. (e) JAGN1-mutated neutrophils (P7) show rapid loss of the mitochondrial membrane potential (Δψmt) upon stimulation with valinomycin, as determined by flow cytometric analysis using the lipophilic cation JC-1 as dye.

Figure 3

A role of JAGN1 in the secretory pathway. (a) Endogenous JAGN1 co-localized mainly with endoplasmic reticulum protein calnexin. HeLa cells were immunostained with anti-JAGN1 antibody (green) and anti-calnexin antibody for visualisation of ER (red). DAPI staining was used to visualize nuclei (blue). Images were acquired by a confocal laser scanning microscope fitted with a 63x objective. Scale bar, 20μm. (b) siRNA-mediated knockdown of JAGN1 leads to significantly (p <0.05) decreased secretion of Gaussia luciferase in comparison to control. For control, scrambled siRNA was used in this experiment. Thapsigargin was used as a positive control for secretion as it induces ER stress. The result shows a ratio between samples averaged over three independent experiments, statistical significance was calculated using a two-tailed Student’s t-test. (c) Determination of JAGN1-protein complex. Tandem affinity purification of N- and C-terminally STREP-HA-tagged JAGN1 protein complexes followed by liquid chromatography mass spectrometry identified several interacting proteins, including members of the COPI complex. (d) Immunoprecipitation of COPA and COPB2 using N- and C-terminally STREP-HA-tagged JAGN1 was performed and confirmed physical interaction between these proteins, consistent with the data shown in (c). (e) Western blotting to study glycosylation pattern of G-CSF-R in patient neutrophils. Neutrophils were isolated from blood from a healthy donor and patients of three families (P13, P2 and P12, respectively). Neutrophils from a healthy donor were also treated with PNGase F to remove glycans. GAPDH or ACTIN served as loading controls in panels (d) and (e), respectively.