The Palmitoyl Transferase Activity of Lecithin Retinol Acyl Transferase

Linlong Xue

Wan Jahng

Deviprasad Gollapalli

Robert Rando

Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, 45 Shattuck Street, Boston, MA 02115

^{To whom correspondence should be addressed. 617−432−1794 (tel), 617−432−0471 (Fax);}ude.dravrah.smh@odnar (email)

Abstract

Lecithin retinol acyl transferase (LRAT) has the essential role of catalyzing the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate all-trans-retinyl esters (tREs). In vitro studies had previously shown that LRAT can also exchange palmitoyl groups between RPE65, a tRE binding protein essential for vision, and tREs. This exchange is likely to be of regulatory significance in the operation of the visual cycle.

In the current study, the substrate specificity of LRAT is explored with palmitoylated amino acids and dipeptides as RPE65 surrogates. Both O and S-substituted palmitoylated analogs are excellent substrates for tLRAT, a readily expressed and readily purified form of LRAT. Using vitamin A as the palmitoyl acceptor, tREs are readily formed. The cognate of these reactions occurs in crude retinal pigment epithelial (RPE) membranes as well. RPE membranes containing LRAT transfer palmitoyl groups from radiolabeled DP*PC to RPE65. Palmitoyl transfer is abolished by preincubation with a specific LRAT antagonist both in membranes and with purified tLRAT. These experiments are consistent with an expanded role for LRAT function as a protein palmitoyl transferase.

Keywords: Lecithin Retinol Acyl Transferase, Acyl Donor, Peptide Substrates, Protein Palmitoylation

Abstract

The phototransduction cascade in vision is initiated by the cis→trans photoisomerization of the protonated 11-cis-retinal Schiff base chromophore of rhodopsin into its all-trans congener (1, 2). The regeneration of rhodopsin after bleaching requires a series of biochemical reactions to resynthesize the 11-cis-retinal chromophore as shown in Scheme 1 (3). The critical trans→cis isomerohydrolase (IMH) reaction minimally requires two known components-lecithin retinol acyl transferase (LRAT) and RPE65. LRAT is necessary and sufficient for the biosynthesis of all-trans-retinyl esters, the substrates for the isomerization reaction (4-6). RPE65 has been determined to be an all-trans-retinyl ester (tRE) binding protein in vitro when purified (7, 8). Based on indirect expression experiments some believe that RPE65 is also an enzyme responsible for the critical trans→cis isomerohydrolase reaction of the visual cycle (9-11). However, this conclusion seems to be in conflict with the fact that purified RPE65 is chemically inert, and thus unable to convert tREs into 11-cis-retinol (8). It is likely that other, possibly unidentified, proteins are essential for biologically relevant isomerization.

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Scheme 1

The Mammalian Visual Cycle

Whatever its precise function, RPE65 is certainly a protein of central importance in visual cycle function (12). It was first discovered as a major protein concentrated in the retinal pigment epithelium (13). Knockout studies of this protein of unknown function in mice revealed it to be necessary for the biosynthesis of 11-cis-retinoids (12). The membrane-associated form of RPE65 (mRPE65) was later shown to stereoselectively bind hydrophobic all-trans retinyl esters with high affinity (7, 14). mRPE65 has been found to be palmitoylated at three cysteine residues: C231, C329, and C330 (15). C330 is completely conserved in the known RPE65 homologs, and C231 is largely conserved except for the highly conservative S/C substitution in zebra fish (16). The hydrophobic palmitoylation modification allows the protein to associate with RPE membranes where isomerization ensues. Whether all mRPE65 is completely triply palmitoylated is unknown. The soluble form of RPE65 (sRPE65) is not triply palmitoylated and binds all-trans-retinol (vitamin A) with a substantially higher affinity than it binds to tREs (15). Importantly, m and sRPE65 are interconverted by LRAT (15). In other words, they are substrates for this enzyme. This surprising result immediately suggests a regulatory loop in which the visual cycle is under the control of the palmitoylation state of RPE65 (15). This can readily be understood when considering what transpires when 11-cis-retinol, the product of IMH processing, builds up. The 11-cis-retinol is esterified by LRAT, using mRPE65 as the palmitoyl donor. Since only mRPE65 is competent to bind tREs so that they can be engaged in the isomerohydrolase reaction, the conversion of mRPE65 to sRPE65 has the net effect of blocking isomerization, and hence regulating the system (15). This regulatory mechanism also requires that RPE65 function is rate limiting in operation of the visual cycle. Independent evidence supports this view. Specific antagonists of mRPE65 inhibit rates of rhodopsin regeneration in vivo in rodents (17) and rhodopsin regeneration rates in mice are dependent on RPE65 levels (18)

There are several outstanding questions concerning the biochemical basis for the respective roles of LRAT and RPE65 in the regulatory switch mechanism. In this mechanism, a palmitoyl thioester of mRPE65 is transferred to vitamin A, suggesting that the latter is a peptidomimetic of lecithin, the standard acyl ester donor for LRAT. In the current studies, we address the substrate specificity of tLRAT, a truncated and readily expressible variant of LRAT (19), with respect to acyl donor specificity. We report that simple acyl cysteine and serine-containing molecules are excellent substrates for tLRAT. Therefore, the enzyme processes both ester and thioester moieties found in simple amino acids and dipeptides. It is also important to establish that LRAT mediated palmitoyl transfer occurs in the biological context of RPE membranes, in addition to occurring with purified proteins. We also provide evidence for the LRAT-mediated transfer of palmitoyl groups to RPE65 in crude membranes derived from bovine RPE. Thus LRAT functions as a protein palmitoyl transferase both in the purified form and in crude membranes.

Abbreviations

BSA	bovine serum albumin
DPPC	L-α-dipalmitoylphosphatidylcholine
DP*PC	[1-^{C]-L-α-dipalmitoyl diphosphatidylcholine}
LRAT	lecithin retinol acyltransferase
tLRAT	truncated lecithin retinol acyltransferase
tRE	all-trans-retinyl ester
RPE	retinal pigment epithelium
TFA	trifluoroacetic acid
DTT	dithiothreitol
EDTA	ethylenediaminetetraaceticacid disodium salt
tRBA	all-trans-retinyl α-bromoacetate
CHAPS	3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate
CHAPSO	3-[(3-Cholamidopropyl-dimethylammoniol-2-hydroxy-1-propane sulfonate
THF	tetrahydrofuran
IEF	isoelectrofocusing
DMSO	dimethyl sulfoxide
SDS-PAGE	sodium dodecylsulfate-polyacrylamide gel electrophoresis

Abbreviations

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