The UBIAD1 prenyltransferase links menaquinone-4 [corrected] synthesis to cholesterol metabolic enzymes

Abstract

Schnyder corneal dystrophy (SCD) is an autosomal dominant disease characterized by germline variants in UBIAD1 introducing missense alterations leading to deposition of cholesterol in the cornea, progressive opacification, and loss of visual acuity. UBIAD1 was recently shown to synthesize menaquinone-4 (MK-4, vitamin K(2) ), but causal mechanisms of SCD are unknown. We report a novel c.864G>A UBIAD1 mutation altering glycine 177 to glutamic acid (p.G177E) in six SCD families, including four families from Finland who share a likely founder mutation. We observed reduced MK-4 synthesis by UBIAD1 altered by SCD mutations p.N102S, p.G177R/E, and p.D112N, and molecular models showed p.G177-mutant UBIAD1 disrupted transmembrane helices and active site residues. We show UBIAD1 interacts with HMGCR and SOAT1, enzymes catalyzing cholesterol synthesis and storage, respectively, using yeast two-hybrid screening and immunoprecipitation. Docking simulations indicate cholesterol binds to UBIAD1 in the substrate-binding cleft and substrate-binding overlaps with GGPP binding, an MK-4 substrate, suggesting potential competition between these metabolites. Impaired MK-4 synthesis is a biochemical defect identified in SCD suggesting UBIAD1 links vitamin K and cholesterol metabolism through physical contact between enzymes and metabolites. Our data suggest a role for endogenous MK-4 in maintaining cornea health and visual acuity.

Figure 1.

Proband corneas and sequencing. A: Family A, a 51 year old female (IV:29, Fig. 2A) with central corneal opacity with crystals, mid peripheral haze, and arcus lipoides. B: Family B, a 64 year old female (IV:19, Fig. 2B) with paracentral crystalline deposits, diffuse corneal haze, and arcus lipoides. C: Family C, a 72 year old female (III:6, Fig. 2C) with central corneal opacification, mid peripheral haze, and arcus lipoides. D: Family D, an 81 year old female (III:11, Fig. 2D) with central corneal opacity and crystals, mid peripheral haze, and arcus lipoides. E: Family JJ, a 73 year old female (II:2, Fig. 2E) with diffuse corneal haze and central disciform opacity with subepithelial crystalline deposits, mid peripheral haze, and peripheral arcus lipoides. A cornea photo of the Family W proband has been published [Köksal et al., 2004]. F: Representative sequences of the n.864 G/A mutation from the Family A proband (top) and an unaffected donor (bottom). Sequences from other probands were similar.

Figure 2.

SCD families with UBIAD1 p.G177E. A-F: SCD Swede-Finn families A-D, Japanese Family JJ, and Turkish Family W, respectively. Circles indicate females; squares, males; filled symbols, affected individuals; horizontal white line in a filled symbol, cornea transplant surgery (penetrating keratoplasty); vertical line, clinically or self-reported negative for SCD but positive for a p.G177E mutation; E1, clinical examination: (+) affected, (−) unaffected; E2, genetic analysis: (+) G>A alteration, (−) wild type; asterisks, examined by J.S.W.

Figure 3.

SCD mutations and the UBIAD1 protein. A: Distribution of SCD family mutations on a linear protein model. Nine of ten predicted TM helices (grey) are in a predicted prenyltransferase domain (horizontal line, bottom) containing 19/19 residues altered in SCD families (one arrow per family), including p.G177E mutations in Swede-Finn (blue arrows) and Japanese and Turkish (green arrows) SCD families. A germline variant, p.S75F, is shown (red arrow). B: UBIAD1 in a lipid bilayer. SCD mutations (black boxes) occur in Loops 1–3 (shaded), including mutation hotspots asparagine 102 and glycine 177 (arrows). A predicted active site based on E.coli menA (dotted line, Weiss et al., 2008) and CRAC and FARM domains (Fredericks et al., 2011) are indicated. Acidic (blue), basic (red), a p.S75F SNP (green), and residues outside the predicted prenyltransferase domain (orange) are indicated. C: Complete conservation of codon p.G177 in 19 species.

Figure 4.

MK-4 biosynthesis by wt and SCD mutant UBIAD1. A: Endogenous and transiently expressed UBIAD1 in HEK293 cells, and WT and SCD mutant UBIAD1 in cell lysates from unaffected (normal) and SCD mutant subjects. Protein amounts were normalized using an UBIAD1-specific antibody. B: MRM chromatograms from LC-APCI-MS/MS analysis of MK-4-d₇ (arrows) converted from PK-d₇ or K3-d₈ in the microsomal fractions of cell lysates from WT and SCD mutant subjects. C: Quantitation of MK-4-d₇ synthesis. Values are means ± SD (n=3) and those significantly different from wt UBIAD1 activity are indicated (*p<0.05, Student’s t-test).

Figure 5.

Molecular modeling and substrate docking simulations. A: 3D model of the native protein showing p.G177 at the top of helix 5 in loop 2 and docked substrates, GGPP and menadione, partly visible behind and to the right of helix 6. Mg²⁺ required to activate the diphosphate for prenyl transfer is shown (green ball). B: Structural changes induced by a p.G177E in silico substitution. C, D: Details of altered interactions (dotted lines) between UBIAD1 active site residues and substrates menadione (green) and GGPP (magenta) in native (panel C) and p.G177E (panel D) proteins. Mg²⁺ (green ball), diphosphate moiety of GGPP (red and orange). E: Overlap of GGPP and cholesterol in the UBIAD1 binding cleft. GGPP (magenta) and menaquinone (green) or cholesterol (cyan) was independently docked to UBIAD1 and substrate positions were compared. Binding sites overlapped as shown, indicating potential competitive occupancy of the substrate binding cleft. UBIAD1 carbon residues (grey), oxygen (red), and the GGPP diphosphate (red/orange) are highlighted. Orientation of substrates is similar to Fig. 5C and D.

Figure 6.

Confirmation of HMGCR and SOAT1 interactions with UBIAD1. A,C: Anti-Myc to examine Flag and Myc IPs after transient transfection with indicated DNAs (left). The expected HMGCR molecular weight (MW, 97 kDa) and markers are indicated (left). B,D: Antibodies against a Flag tag were used to examine Flag and Myc IPs after transient transfection with the indicated DNAs (left). The expected UBIAD1 MW was 37 kDa. E: HEK293T cells expressing both 3xFlag-UBIAD1 and Myc-HMGCR were stained with mouse anti-Flag and rabbit Anti-Myc antibodies and secondary antibodies conjugated to either AlexaFluor488 (anti-mouse) or AlexaFluor568 (anti-rabbit). Representative images are shown; untransfected cells showed no signal (unpublished data).