Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT^−/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT^−/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT^−/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT^−/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.     

Hepatic stellate cell lipid droplets: A specialized lipid droplet for retinoid storage

The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease.     

Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage

Hepatic stellate cell (HSC) lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT) mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS) based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP) mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i) increased expression of typical markers of HSC activation; (ii) decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT); (iii) decreased triglyceride levels; (iv) increased expression of genes associated with lipid catabolism; and (v) an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1. CONCLUSION: Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.     

Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT)

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2-3-fold) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3-4-fold) in the level of cytosolic retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary retinol primarily as free retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these (chylomicron) retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:retinol acyltransferase.     

Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions

It is now well documented that lecithin-retinol acyltransferase (LRAT) is the physiologically important enzyme activity involved in the esterification of retinol in the liver. However, no information regarding the cellular distribution of this enzyme in the liver is presently available. This study characterizes the distribution of LRAT activity in the different types of rat liver cells. Purified preparations of isolated parenchymal, fat-storing, and Kupffer + endothelial cells were isolated from rat livers and the LRAT activity present in microsomes prepared from each of these cell fractions was determined. The fat-storing cells were found to contain the highest level of LRAT specific activity (383 +/- 54 pmol retinyl ester formed min-1.mg-1 versus 163 +/- 22 pmol retinyl ester formed min-1.mg-1 for whole liver microsomes). The level of LRAT specific activity in parenchymal cell microsomes (158 +/- 53 pmol retinyl ester formed min-1.mg-1) was very similar to LRAT levels in whole liver microsomes. The Kuppfer + endothelial cell microsome fractions were found to contain LRAT, at low levels of activity. These results indicate that the fat-storing cells are very enriched in LRAT but the parenchymal cells also posses significant levels of LRAT activity.     

Genetically modified mouse models to study hepatic neutral lipid mobilization

Excessive accumulation of triacylglycerol is the common denominator of a wide range of clinical pathologies of liver diseases, termed non-alcoholic fatty liver disease. Such excessive triacylglycerol deposition in the liver is also referred to as hepatic steatosis. Although liver steatosis often resolves over time, it eventually progresses to steatohepatitis, liver fibrosis and cirrhosis, with associated complications, including liver failure, hepatocellular carcinoma and ultimately death of affected individuals. From the disease etiology it is obvious that a tight regulation between lipid uptake, triacylglycerol synthesis, hydrolysis, secretion and fatty acid oxidation is required to prevent triacylglycerol deposition in the liver. In addition to triacylglycerol, also a tight control of other neutral lipid ester classes, i.e. cholesteryl esters and retinyl esters, is crucial for the maintenance of a healthy liver. Excessive cholesteryl ester accumulation is a hallmark of cholesteryl ester storage disease or Wolman disease, which is associated with premature death. The loss of hepatic vitamin A stores (retinyl ester stores of hepatic stellate cells) is incidental to the onset of liver fibrosis. Importantly, this more advanced stage of liver disease usually does not resolve but progresses to life threatening stages, i.e. liver cirrhosis and cancer. Therefore, understanding the enzymes and pathways that mobilize hepatic neutral lipid esters is crucial for the development of strategies and therapies to ameliorate pathophysiological conditions associated with derangements of hepatic neutral lipid ester stores, including liver steatosis, steatohepatitis, and fibrosis. This review highlights the physiological roles of enzymes governing the mobilization of neutral lipid esters at different sites in liver cells, including cytosolic lipid droplets, endoplasmic reticulum, and lysosomes. This article is part of a Special Issue entitled Molecular Basis of Disease: Animal models in liver disease.     

Vitamin A status regulates hepatic lecithin: retinol acyltransferase activity in rats

The activity of lecithin:retinol acyltransferase (LRAT) was determined in microsomes from the liver and small intestine of rats with differing vitamin A status. In animals depleted of retinol, as judged by undetectable liver vitamin A stores and low plasma retinol concentrations, hepatic LRAT activity was almost undetectable, whether assayed with retinol bound to cellular retinol-binding protein or solvent-dispersed retinol. In contrast, neither the activity of intestinal LRAT nor that of acyl-CoA:retinol acyltransferase in either liver or intestine differed from that of vitamin A-adequate rats. During the course of vitamin A depletion, liver LRAT activity fell progressively, nearly in parallel to the decrease in plasma retinol concentration. Oral repletion of vitamin A-depleted rats with 0.8 mg of retinol resulted in a very rapid restoration of plasma retinol concentration and full recovery of hepatic LRAT activity within 24 h, together with deposition of retinyl ester in the liver. These data strongly implicate LRAT activity in liver as responsible for the storage of hepatic retinyl esters. Retention of the intestine's capacity to esterify retinol during vitamin A deficiency provides a mechanism for capture of dietary vitamin A, while reduced hepatic LRAT activity may function to redirect retinol in liver from storage to other metabolic pathways.     

KIAA1363 affects retinyl ester turnover in cultured murine and human hepatic stellate cells

Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, we identified KIAA1363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase. We show that KIAA1363 is expressed in the liver, mainly in HSCs, and exhibits RE hydrolase activity at neutral pH. Accordingly, addition of the KIAA1363-specific inhibitor JW480 largely reduced RE hydrolase activity in lysates of cultured murine and human HSCs. Furthermore, cell fractionation experiments and confocal microscopy studies showed that KIAA1363 localizes to the endoplasmic reticulum. We demonstrate that overexpression of KIAA1363 in cells led to lower cellular RE content after a retinol loading period. Conversely, pharmacological inhibition or shRNA-mediated silencing of KIAA1363 expression in cultured murine and human HSCs attenuated RE degradation. Together, our data suggest that KIAA1363 affects vitamin A metabolism of HSCs by hydrolyzing REs at the endoplasmic reticulum, thereby counteracting retinol esterification and RE storage in lipid droplets.     

Expression of carboxylesterase and lipase genes in rat liver cell-types

Approximately 80% of the body vitamin A is stored in liver stellate cells with in the lipid droplets as retinyl esters. In low vitamin A status or after liver injury, stellate cells are depleted of the stored retinyl esters by their hydrolysis to retinol. However, the identity of retinyl ester hydrolase(s) expressed in stellate cells is unknown. The expression of carboxylesterase and lipase genes in purified liver cell-types was investigated by real-time PCR. We found that six carboxylesterase and hepatic lipase genes were expressed in hepatocytes. Adipose triglyceride lipase was expressed in Kupffer cells, stellate cells and endothelial cells. Lipoprotein lipase expression was detected in Kupffer cells and stellate cells. As a function of stellate cell activation, expression of adipose triglyceride lipase decreased by twofold and lipoprotein lipase increased by 32-fold suggesting that it may play a role in retinol ester hydrolysis during stellate cell activation.     

The molecular basis of retinoid absorption: a genetic dissection

The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acyl-CoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.     

Vitamin A metabolism in the human intestinal Caco-2 cell line

The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinoids in health and disease: A role for hepatic stellate cells in affecting retinoid levels

Vitamin A (retinol) is important for normal growth, vision and reproduction. It has a role in the immune response and the development of metabolic syndrome. Most of the retinol present in the body is stored as retinyl esters within lipid droplets in hepatic stellate cells (HSCs). In case of liver damage, HSCs release large amounts of stored retinol, which is partially converted to retinoic acid (RA). This surge of RA can mediate the immune response and enhance the regeneration of the liver. If the damage persists activated HSCs change into myofibroblast-like cells producing extracellular matrix, which increases the chance of tumorigenesis to occur. RA has been shown to decrease proliferation and metastasis of hepatocellular carcinoma. The levels of RA and RA signaling are influenced by the possibility to esterify retinol towards retinyl esters. This suggests a complex regulation between different retinoids, with an important regulatory role for HSCs.     

Topology and membrane association of lecithin: retinol acyltransferase

Fatty acid retinyl esters are the storage form of vitamin A (all-trans-retinol) and serve as metabolic intermediates in the formation of the visual chromophore 11-cis-retinal. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, acts by transferring an acyl group from the sn-1 position of phosphatidylcholine to retinol. To define the membrane association and localization of LRAT, we produced an LRAT-specific monoclonal antibody, which we used to study enzyme partition under different experimental conditions. Furthermore, we examined the membrane topology of LRAT through an N-linked glycosylation scanning approach and protease protection assays. We show that LRAT is localized to the membrane of the endoplasmic reticulum (ER) and assumes a single membrane-spanning topology with an N-terminal cytoplasmic/C-terminal luminal orientation. In eukaryotic cells, the C-terminal transmembrane domain is essential for the activity and ER membrane targeting of LRAT. In contrast, the N-terminal hydrophobic region is not required for ER membrane targeting or enzymatic activity, and its amino acid sequence is not conserved in other species examined. We present experimental evidence of the topology and subcellular localization of LRAT, a critical enzyme in vitamin A metabolism.     

Retinyl esters are the substrate for isomerohydrolase

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.     

All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle

The identification of the critical enzyme(s) that carries out the trans to cis isomerization producing 11-cis-retinol during the operation of the visual cycle remains elusive. Confusion exists in the literature as to the exact nature of the isomerization substrate. At issue is whether it is an all-trans-retinyl ester or all-trans-retinol (vitamin A). As both putative substrates interconvert rapidly in retinal pigment epithelial membranes, the choice of substrate can be ambiguous. The two enzymes that effect interconversion of all-trans-retinol and all-trans-retinyl esters are lecithin retinol acyl transferase (LRAT) and retinyl ester hydrolase (REH). The retinyl ester or all-trans-retinol pools are radioactively labeled separately in the presence of inhibitors of LRAT and REH, effectively preventing their interconversion. Pulse-chase experiments unambiguously demonstrate that all-trans-retinyl esters, and not all-trans-retinol, are the precursors of 11-cis-retinol. When the all-trans-retinyl ester pool is radioactively labeled, the resulting 11-cis-retinol is labeled with the same specific activity as the precursor ester. The converse is true with vitamin A. These data unambiguously establish all-trans-retinyl esters as the precursors of 11-cis-retinol.     

Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver

Lecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat-/-) mouse. Despite the expression of LRAT in multiple tissues, the Lrat-/- mouse develops normally. The histological analysis and electron microscopy of the retina for 6-8-week-old Lrat-/- mice revealed that the rod outer segments are approximately 35% shorter than those of Lrat+/+ mice, whereas other neuronal layers appear normal. Lrat-/- mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat-/- mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation.     

Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells

Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue.Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL.Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels.In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.     

Two histidine residues are essential for catalysis by lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) is a novel membrane bound enzyme that catalyzes the formation of retinyl esters from vitamin A and lecithin. The enzyme is both essential for vision and for the general mobilization of vitamin A. The sequence of LRAT defines it as a novel enzyme unrelated to any other protein of known function. LRAT possesses a catalytically essential active site cysteine residue. The enzyme also contains six histidine residues. It is shown here that two of these residues (H57 and H163) are essential for catalysis. A mechanistic hypothesis is presented to account for these observations.     

An Acyl-covalent Enzyme Intermediate of Lecithin:Retinol Acyltransferase

Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromophore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys¹⁶¹ residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis.