Comparison between the enzymatic activity, structure and substrate binding of mouse and human lecithin retinol acyltransferase

Highlights

• The enzymatic activity of mouse truncated LRAT (mtLRAT) is 2.7-fold lower than that of human tLRAT (htLRAT).

• mtLRAT proteins are less thermostable than htLRAT proteins.

• The affinity binding of mtLRAT for its substrate is 3.9-fold lower than that of htLRAT.

• The enzymatic activity of the P173L-htLRAT mutant is 6.3-fold lower compared to that of htLRAT.

Abstract

Lecithin retinol acyltransferase (LRAT) is involved in the visual cycle where it catalyzes the formation of all-trans retinyl ester. The mouse animal model has been widely used to study LRAT. Primary sequence alignment shows 80% identity and 90% similarity between human and mouse LRAT. However, human LRAT has a proline at position 173 (hLRAT (P173)) while an arginine can be found at this position for the mouse protein (mLRAT (R173)). Moreover, residue 173 is important for the human protein since a substitution mutation of this residue to a leucine (P173L-hLRAT) caused night blindness in a patient. The present study was thus undertaken to determine whether mouse and human LRAT have a similar enzymatic activity, structure and substrate binding affinity using a truncated form of LRAT (tLRAT). The enzymatic activity and binding affinity to the substrate, all-trans retinol, of mtLRAT (R173) were found to be 2.7- and 3.9-fold lower, respectively, than that of htLRAT (P173). Moreover, the enzymatic activity of P173L-htLRAT is 6.3-fold lower compared to that of htLRAT (P173). Furthermore, a significant difference was observed between the intrinsic fluorescence emission as well as between the circular dichroism spectra of mtLRAT (R173) and htLRAT (P173). In addition, mtLRAT proteins are less thermostable than htLRAT proteins, which suggests that structural differences exist between the mouse and human proteins. Altogether, these data strongly suggest that the much lower catalytic activity of mtLRAT (R173) compared to that of htLRAT (P173) mostly results from differences between their structure, predominantly revealed by their dissimilar thermal stability, as well as their efficiency to bind all-trans retinol. Therefore, conclusions regarding the behavior of human LRAT based on measurements performed with mouse LRAT must be made with caution. Also, the much lower enzymatic activity of P173L-htLRAT compared to that of htLRAT (P173) might explain the night blindness of a patient carrying this mutation.

Introduction

Lecithin: retinol acyltransferase (LRAT; UniProtKB AAD13529) is an important enzyme of the visual cycle. It catalyzes the esterification of all-trans retinol into all-trans retinyl ester, which is a key step in the regeneration of the chromophore of the visual pigment rhodopsin [1,2]. LRAT can be found in different tissues including the retina, intestines, liver and testis [[3], [4], [5]]. The primary sequence of human LRAT (hLRAT) consists of 230 residues (Fig. 1A), with a calculated mass of 25.3 kDa [6]. Analysis of this sequence suggests the presence of potential transmembrane N- and C-terminal hydrophobic segments between residues 9–31 and 195–222, respectively [[6], [7], [8]]. We and others have thus produced a recombinant truncated human LRAT (htLRAT) (residues 31–196) to obtain a soluble protein [9,10]. The residues essential for the catalytic activity of LRAT (H60, H72, Y154 and C161, Fig. 1A) [9,11,12] are present in htLRAT, whose enzymatic activity has been characterized in detail [13]. LRAT hydrolyzes the sn-1 fatty acyl chain of phospholipids, which serves to acylate its C161; this acyl group is then transferred to all-trans retinol to form all-trans retinyl ester [3,12,14,15]. Mutations in LRAT lead either to a modification of its open reading frame, to a premature termination of its synthesis [16,17] or to amino acid substitutions (Fig. 1A), which include E14L, Y61D, A106T [18], R109L [19], R109C/R190H [20], S175R [21] and P173L [22]. A complete loss of LRAT enzymatic activity has been identified as the cause of the disease resulting from the S175R mutation [21,23]. The unusual disease generated from mutation P173L causes a decrease in night vision during infancy and a loss of the visual field a little before 60 years old [22]. In contrast, mutations E14L, Y61D, A106T, R109L, R109C/R190H and S175R lead to severe vision loss during infancy, which quickly evolves to blindness [18].

Mice have been extensively used to study LRAT (for a review, see Refs. [[24], [25], [26]]). For instance, the function of LRAT was first determined using mouse LRAT (mLRAT) and mice models have been used to understand eye diseases caused by LRAT mutations [27,28]. As shown in Fig. 1A, the primary sequence of mLRAT (231 residues) has 80% identity and 90% similarity with that of hLRAT. In particular, an arginine is present at position 173 in mLRAT (mLRAT (R173)) whereas a proline can be found at this position in hLRAT (hLRAT (P173)). There is a large structural and chemical difference between these two amino acids. In addition, mutation P173L leads to a mild form of retinitis pigmentosa [22]. One could thus postulate that the enzymatic activity of mLRAT (R173) is different from that of hLRAT (P173), which also differs from that of the P173L-htLRAT mutant.

This study was thus undertaken to determine whether differences in the enzymatic activity, structure and binding affinity for all-trans retinol exist between htLRAT (P173) and mtLRAT (R173). Moreover, the effect of changing the proline in the human protein by an arginine (P173R-htLRAT) and, conversely, of modifying the arginine in the mouse protein for a proline (R173P-mtLRAT) on their enzymatic activity and thermal stability was also assayed. This was also done for the P173L-htLRAT mutant leading to photoreceptor degeneration. Fluorescence spectroscopy and circular dichroism were also used to characterize mouse and human tLRAT proteins and to provide explanations for the discrepancy between their catalytic activity by comparing the environment of their W158, their secondary structure and thermal stability as well as their ability to bind all-trans retinol.