A disease-associated missense mutation in CYP4F3 affects the metabolism of leukotriene B4 via disruption of electron transfer

Abstract

Background: Cytochrome P450 4F3 (CYP4F3) is an ω-hydroxylase that oxidizes leukotriene B4 (LTB4), prostaglandins, and fatty acid epoxides. LTB4 is synthesized by leukocytes and acts as a chemoattractant for neutrophils, making it an essential component of the innate immune system. Recently, involvement of the LTB4 pathway was reported in various immunological disorders such as asthma, arthritis, and inflammatory bowel disease. We report a 26-year-old female with a complex immune phenotype, mainly marked by exhaustion, muscle weakness, and inflammation-related conditions. The molecular cause is unknown, and symptoms have been aggravating over the years.

Methods: Whole exome sequencing was performed and validated; flow cytometry and enzyme-linked immunosorbent assay were used to describe patient's phenotype. Function and impact of the mutation were investigated using molecular analysis: co-immunoprecipitation, western blot, and enzyme-linked immunosorbent assay. Capillary electrophoresis with ultraviolet detection was used to detect LTB4 and its metabolite and in silico modelling provided structural information.

Results: We present the first report of a patient with a heterozygous de novo missense mutation c.C1123 > G;p.L375V in CYP4F3 that severely impairs its activity by 50% (P < 0.0001), leading to reduced metabolization of the pro-inflammatory LTB4. Systemic LTB4 levels (1034.0 ± 75.9 pg/mL) are significantly increased compared with healthy subjects (305.6 ± 57.0 pg/mL, P < 0.001), and immune phenotyping shows increased total CD19+ CD27- naive B cells (25%) and decreased total CD19+ CD27+ IgD- switched memory B cells (19%). The mutant CYP4F3 protein is stable and binding with its electron donors POR and Cytb5 is unaffected (P > 0.9 for both co-immunoprecipitation with POR and Cytb5). In silico modelling of CYP4F3 in complex with POR and Cytb5 suggests that the loss of catalytic activity of the mutant CYP4F3 is explained by a disruption of an α-helix that is crucial for the electron shuffling between the electron carriers and CYP4F3. Interestingly, zileuton still inhibits ex vivo LTB4 production in patient's whole blood to 2% of control (P < 0.0001), while montelukast and fluticasone do not (99% and 114% of control, respectively).

Conclusions: A point mutation in the catalytic domain of CYP4F3 is associated with high leukotriene B4 plasma levels and features of a more naive adaptive immune response. Our data provide evidence for the pathogenicity of the CYP4F3 variant as a cause for the observed clinical features in the patient. Inhibitors of the LTB4 pathway such as zileuton show promising effects in blocking LTB4 production and might be used as a future treatment strategy.

Keywords: CYP4F3; Cytb5; Exhaustion; LTB4; Muscle weakness; POR.

Figure 1

Heterozygous de novo CYP4F3 L375V mutation identified via whole exome sequencing. (A) PBMCs isolated from healthy controls and the index patient were stained and analysed by flow cytometry. Total monocyte cell numbers as a percentage of total cells. Total B cell numbers (CD19+) as a percentage of total cells. Total naive B cell numbers (CD19+ CD27−) as a percentage of total B cells. Total switched memory B cell numbers (CD19+ CD27+ IgD−) as a percentage of total B cells. Total T cell numbers (CD3+) as a percentage of total cells. Total naive CD4+ T cell numbers (CD4+ CCR7+ CD45RA+) as a percentage of total CD4+ T cells. Total naive CD8+ T cell numbers (CD8+ CCR7+ CD45RA+) as a percentage of total CD8+ T cells. Average ±SEM. (B) Pedigree of a family with segregation of the L375V mutant allele. Patient is denoted with a heterozygous de novo L375V mutation in black. White square means male; white circle means female. (C) Sanger sequencing confirming heterozygous L375V mutation in the patient's genomic DNA, while both parents have WT alleles. (D) Multiple sequencing alignment of CYP4F3 displaying conservation of the 351–400 region encompassing the L375V mutation from different organisms. Identical amino acids compared with the Homo sapiens sequence are indicated by dashes; spaces indicate gaps in the alignment. Data were retrieved from Homologene (NCBI) and Ensembl.

Figure 2

L375V mutation reduces LTB4 ω‐hydroxylation capacity. (A) Quantification of LTB4 levels in plasma from patient and healthy subjects. Each data point represents a different sample. Average ±SEM, one‐way ANOVA, ***P < 0.001. (B) CE‐UV electropherogram of postnuclear supernatant containing CYP4F3 WT or L375V mutant, incubated with 20 μM LTB4 for 60 min at 37°C. BGE for CE‐UV: borate buffer with 12.5 mM SDS at pH 8.3, and detection wavelength is 270 nm. (C) Quantification of CE‐UV detection of 20OH‐LTB4 conversion. WT or L375V CYP4F3 were incubated with LTB4 for 60 min at 37°C (n = 6). Average ±SEM, paired t‐test, *P < 0.05. (D) Quantification of CE‐UV detection of 20OH‐LTB4 conversion. WT or mutant CYP4F3 were incubated with LTB4 for 200 min at 37°C (n = 2). Average ±SEM, paired t‐test, **P < 0.01. (E) Western blot showing protein expression of Flag‐tagged WT and mutant CYP4F3. (F) Quantification of 20OH‐LTB4 formation via CE‐UV detection after incubating postnuclear supernatant containing WT or mutant CYP4F3 with LTB4 for time periods ranging from 0 to 200 min (n = 2). Average ±SEM, nonlinear regression, ****P < 0.0001.

Figure 3

The CYP4F3 interaction with electron transfer partners is unaffected by the L375V mutation. (A) Homology model of CYP4F3 (green) in complex with the FMN‐domain of POR (pink). The substrate binding site of CYP4F3 is depicted in yellow and the heme group is visualized. The L375V mutation (purple) is located in the binding interface of CYP4F3 with POR. Flavin rings of POR are illustrated as well. (B) Homology model of CYP4F3 (green) in complex with Cytb5 (blue). The substrate binding site of CYP4F3 is depicted in yellow and the heme group is visualized. The L375V mutation (purple) is located in the binding interface of CYP4F3 with Cytb5. The heme group of Cytb5 is illustrated in blue. (C) Schematic diagram of HA‐tagged proteins used for the co‐IP assays. Proteins were precipitated using Pierce™ HA‐Tag co‐IP Kit (ThermoFisher scientific). (D) Co‐immunoprecipitated proteins of pulldown with HA‐tagged POR or Cytb5 were detected using western blot. Anti‐Flag antibody was used to detect WT and L375V mutant CYP4F3 (±66 kDa). The total protein fraction before co‐IP is depicted as input, while IP visualizes the immunoprecipitated fraction containing POR or Cytb5 and its bound proteins. (E) Quantification of co‐IP for CYP4F3A with POR (n = 6) and Cytb5 (n = 3). Average ±SEM, one‐way ANOVA.

Figure 4

The L375V mutation destabilizes the α‐helix of the electron transfer path. (A) In silico model visualizing CYP4F3 (green) in complex with both POR (pink) and cytochrome b5 (blue) during the electron shuffling process. The heme group is visualized, and the L375V mutation is depicted in purple. The flavin rings of POR and the heme group of Cytb5 (blue) are visualized as well. The residues involved in electron shuffling, F461 and Q471 (orange), and the co‐evolving residues that are forming a triad with L375V, T382 and K479 (light blue) are in close proximity. (B) Co‐evolutionary analysis comparing both the contact map based on our homology model and the co‐evolving residues. Evolutionarily coupled residues T382 and K479 are indicated in red. (C) Multiple sequence alignment of CYP4F3 displaying conservation of the surrounding regions of L375, T382, and K479 residues in CYP4F3 from different organisms. Identical amino acids compared with the Homo sapiens sequence are indicated by dashes; spaces indicate gaps in the alignment. Two regions are separated by dots.

Figure 5

Zileuton inhibits LTB4 production in patient's blood. (A) Overview of LTB4 metabolism in leukocytes, showing sites of inhibition and potential drugs. LTA4H: LTA4 hydrolase, LTC4S: LTC4 synthase, BLT1R: LTB4 receptor 1. (B) Novel ex vivo assay allowing efficient screening of potential drugs targeting LTB4 synthesis. Whole blood was incubated with 1 μM montelukast, 10 μM zileuton, or 100 pM fluticasone to mimic physiological blood concentrations under treatment. LTB4 synthesis is induced by addition of 30 μM calcium ionophore A23187 at 37°C for 30 min, and LTB4 levels were analysed with the LTB4 ELISA (ThermoFisher Scientific) (n = 4). Average ±SEM, one‐way ANOVA, ****P < 0.0001.