The weak enzymatic activity of truncated lecithin retinol acyltransferase (LRAT) mutants cannot be explained by their affinity for all-trans retinol

Abstract

Purpose : Lecithin retinol acyltransferase (LRAT) is an enzyme responsible for transforming all-trans retinol (ROH) into all-trans retinyl ester. Mutations found in LRAT are responsible for severe degenerative eye diseases. These mutations cause an almost complete loss of activity in a truncated LRAT, without its hydrophobic N- and C-terminal segments (tLRAT). We verified the hypothesis that this lack of activity observed for the tLRAT mutants could be explained by a deficiency in their binding of ROH using fluorescence spectroscopy.

Methods : tLRAT and its mutants were overexpressed using a bacterial expression system and then purified using affinity chromatography. The binding of ROH by tLRAT and its mutants was measured using fluorescence spectroscopy. The fitting of the curve of the fluorescence intensity as a function of ROH concentration using a mathematical model allowed to calculate the dissociation constant of the binding of ROH to tLRAT and its mutants.

Results : An important increase in the fluorescence intensity of ROH was observed in presence of tLRAT and all its mutants. A value of 34 ± 12 nM was obtained for the dissociation constant of the binding of ROH to native tLRAT. Surprisingly, only the mutant P173L–tLRAT showed a dissociation constant whose value (250 ± 70 nM) differed significantly from that of native tLRAT.

Conclusions : These results suggest that all tLRAT mutants bind ROH as efficiently as native tLRAT, except for mutant P173L-tLRAT. The efficiency of ROH binding to LRAT is therefore not responsible for the large decrease in the activity of this enzyme.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.