Lecithin:Retinol Acyltransferase: A Key Enzyme Involved in the Retinoid (visual) Cycle

Abstract

Lecithin:retinol acyltransferase (LRAT) catalyzes the acyl transfer from the sn-1 position of phosphatidylcholine (PC) to all-trans-retinol, creating fatty acid retinyl esters (palmitoyl, stearoyl, and some unsaturated derivatives). In the eye, these retinyl esters are substrates for the 65 kDa retinoid isomerase (RPE65). LRAT is well characterized biochemically, and recent structural data from closely related family members of the NlpC/P60 superfamily and a chimeric protein have established its catalytic mechanism. Mutations in the LRAT gene are responsible for approximately 1% of reported cases of Leber congenital amaurosis (LCA). Lack of functional LRAT, expressed in the retinal pigmented epithelium (RPE), results in loss of the visual chromophore and photoreceptor degeneration. LCA is a rare hereditary retinal dystrophy with an early onset associated with mutations in one of 21 known genes. Protocols have been devised to identify therapeutics that compensate for mutations in RPE65, also associated with LCA. The same protocols can be adapted to combat dystrophies associated with LRAT. Improvement in the visual function of clinical recipients of therapy with recombinant adeno-associated virus (rAAV) vectors incorporating the RPE65 gene provides a proof of concept for LRAT, which functions in the same cell type and metabolic pathway as RPE65. In parallel, a clinical trial that employs oral 9-cis-retinyl acetate to replace the missing chromophore in RPE65 and LRAT causative disease has proven to be effective and free of adverse effects. This article summarizes the biochemistry of LRAT and examines chromophore replacement as a treatment for LCA caused by LRAT mutations.

Figure 1

Estimated LCA prevalence associated with currently identified genes. LRAT is responsible for <1% of known LCA cases, most of which lack a causative gene. CEP290 and GUCY2D are the most frequently mutated genes in humans with LCA.

Figure 2

Schematics of the retinoid cycle and LRAT enzymatic reaction. (A) Retinoid cycle displaying the enzymatic conversion of all-trans-retinol (atROL) to 11-cis-retinal (11-cis-RAL). Absorption of a photon of light (hv) by the visual pigment (11-cis-RAL-opsin) causes isomerization of the active state of rhodopsin (atRAL-opsin). Hydrolysis of the Schiff base linkage then releases free all-trans-retinal (atRAL), which is subsequently reduced to atROL and transported across the interphotoreceptor matrix where it is esterified by LRAT in the RPE. All-trans-retinol can also be absorbed from the bloodstream via STRA6, providing a substrate for LRAT. (B) LRAT functions as an acyltransferase with sn-1 specificity, transferring an acyl moiety from phosphatidylcholine (PC) to the all-trans-retinol substrate. For more chemical and biochemical details, see refs and .

Figure 3

LRAT exons overlaid with known mutations. (A) LRAT exons mapped to chromosome 4, with known mutations associated with LCA. (B) Sequences of human HRASLS3, HRASLS3/LRAT, and LRAT chimera were aligned with T-Coffee multiple-sequence alignment available at the EMBL-EBI server. Both the conserved His residues and the six-amino acid stretch containing the catalytic Cys residue are colored green. The LRAT-specific domain is highlighted in teal. Point mutations associated with LCA are highlighted in red. (C) Two-dimensional model of an HRASLS3/LRAT protomer showing the sites of naturally occurring mutations associated with LCA. Residues in HRASLS3/LRAT occurring downstream of the arrowhead (→) are hypothetical due to truncation of the C-terminal α helix to promote solubility. Point mutations associated with LCA are highlighted in gray. The model is based on the crystal structure of the chimeric protein.

Figure 4

Membrane topology of a HRASLS3/LRAT chimera with hydrophobic exposed active sites. (A) Ribbon representation of a HRASLS3/LRAT chimera viewed in parallel with a plasma membrane. Red and blue ribbons represent single protomers. Theoretical placement of the C-terminal transmembrane α helices (TMH) spanning the lipid membrane is represented by cylinders. (B) Two perpendicular views from underneath dimeric HRASLS3/LRAT. The bottom view presents a hydrophobic surface map with the proposed membrane interaction surface colored red. Surfaces of the molecules are colored according to their hydrophobicity. Red corresponds to hydrophobic residues that largely comprise the LRAT-specific domain and membrane-interacting surface. (C) Electron density of the acylated form of HRASLS3/LRAT embedded within the hydrophobic active site. The acyl moiety is heptanoic acid bound to Cys-125. Gray mesh represents a 2.2 Å resolution σA-weighted 2F_o − F_c electron density map contoured at 1.6σ. The green mesh represents the unbiased σA-weighted F_o − F_c electron density map contoured at 3.5σ. The figures are adapted from the crystal structure of the chimeric protein.

Figure 5

LRAT knockout murine models lack functional retinoids and display photoreceptor degradation. (A) Retinoids were extracted from whole mouse eye and separated by normal phase high-performance liquid chromatography. The peaks shown correspond to the following retinoids: (1) 11-cis- and 13-cis-retinyl esters, (2) all-trans-retinyl esters, (3) 4′-syn- and anti-all-trans-retinal oximes, (4) 3′-syn- and anti-11-cis-retinal oximes, and (5) all-trans-retinol. The chromatogram was generated as described by Batten et al. with permission from the American Society for Biochemistry and Molecular Biology. Mice were dark adapted for 48 h prior to ocular extraction. (B) P28 and P42 WT and Lrat^−/− mutant mouse retinas were stained for cone pigments: middle- and long-wavelength sensitive (M/L) opsin and short-wavelength (S) opsin are located only within cone cells. A perinuclear ring (white arrows) indicates the endoplasmic reticulum where M/L opsin is synthesized. The absence of these opsins in Lrat^−/− models indicates degeneration of functional cone outer segments. Abbreviations: OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. This figure is adapted from ref with approval from The Association for Research Vision and Ophthalmology.