Role of sn-2 Acyl Group of Phosphatidyl Choline in Determining the Positional Specificity of Lecithin-Cholesterol Acyltransferase

Subbaiah, Papasani V.; Liu, Ming; Paltauf, Fritz

doi:10.1021/bi00249a012

Cited by 42 publications

(46 citation statements)

References 33 publications

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“…These results are similar to the effect of ceramide on sPLA 2 IIa hydrolysis of PE/ PS mixture, where the release of 20:4 and 18:2 was stimulated more than the release of 18:1 (13). It may be pointed out that in the present study, the formation of 16:0 CE was greater than expected from the percent of 16:0-16:0 PC in the mixture (25%), although LCAT is not known to be specific for this PC species (32) (25,34). While these factors resulted in the formation of higher than expected percent of 16:0 CE, the presence of ceramide significantly decreased 16:0 CE synthesis, suggesting a specific inhibitory effect of ceramide on the synthesis of saturated CE.…”

Section: Effect Of Ceramide In Presence Of Sm-results Incontrasting

confidence: 55%

Regulation of the Activity and Fatty Acid Specificity of Lecithin-Cholesterol Acyltransferase by Sphingomyelin and Its Metabolites, Ceramide and Ceramide Phosphate

2006

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Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids were similar on the phospholipase A and cholesterol esterification reactions of the enzyme, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Ceramide incorporation into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response. Abbreviations usedApo : apolipoprotein; CE: cholesteryl ester; FC: free (unesterified) cholesterol; HDL: high density lipoproteins; LCAT: lecithin-cholesterol acyltransferase; LDL: low density lipoproteins; PC: phosphatidylcholine; PLA: phospholipase A; SM: sphingomyelin; SMase: sphingomyelinase Sphingomyelin (SM) is one of the most abundant phospholipids in cell membranes and lipoproteins, constituting up to 30% of certain lipoprotein fractions (1,2). Although its role as a structural component of membranes, and as a precursor of signaling molecules has been well recognized, its function in plasma lipoproteins has received less attention. Recent studies show that plasma SM may be an independent risk factor for atherosclerosis (3), and that a reduction in SM levels by myriocin treatment reduces atherosclerosis in apo E deficient mice (4,5). While the exact mechanism of the proatherogenic effect of SM is unknown, one proposed mechanism involves the generation of ceramide in LDL by the action of arterial SMase, which causes † Supported by a grant from National Institutes of Health, HL 68585. increased retention, aggregation and oxidation of LDL, with subsequent uptake by the scavenger receptors of the macrophage (3). The formation of large amounts of ceramide in the plasma compartment is unlikely because of the...

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Section: Effect Of Ceramide In Presence Of Sm-results Incontrasting

confidence: 55%

Regulation of the Activity and Fatty Acid Specificity of Lecithin-Cholesterol Acyltransferase by Sphingomyelin and Its Metabolites, Ceramide and Ceramide Phosphate

2006

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“…Although hLCAT is generally known to be specific for the sn-2 position of PC, hLCAT is able to transfer a significant percentage of the sn-1 position of the acyl group of certain PC species to cholesterol (13). Subbaiah and colleagues (13) reported that the contribution of the sn-1 acyl group from various 1-palmitoyl-2-acyl PCs for cholesteryl ester synthesis is 1.0% from 1,2-dipalmitoyl-PC, 1.4% from 1-palmitoyl-2-eicosatrinoyl-PC, 7.3% from POPC, 47% from 1-palmitoyl-2-eicosapentaenoyl-PC, 49.9% from PAPC, 54.9% from PDPC, and 72.3% from 1-palmitoyl-2-stearoyl-PC. They concluded that the positional specificity of hLCAT is affected by the chain length of the sn-2 acyl group rather than the carbon number or the number and position of double bonds of the acyl group.…”

Section: Discussionmentioning

confidence: 99%

Positional specificity of lysosomal phospholipase A2

Abe

Hiraoka

Shayman

2006

Journal of Lipid Research

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Lysosomal phospholipase A 2 (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A 1 and phospholipase A 2 activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.-Abe, A., M. Hiraoka, and J. A. Shayman. Positional specificity of lysosomal phospholipase A 2 .

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“…If reesterification of lyso-PC with the released sn-2 fatty acid resulted in regeneration of PC, as described previously (31,32), then PLA 2 activity would be underestimated. Finally, if hLCAT uses some sn-1 fatty acid when it encounters polyunsaturated PC species such as PAPC, PLA 2 activity would also be underestimated (33). Any combination of these possibilities could explain the relatively lower PLA 2 activities.…”

Section: Discussionmentioning

confidence: 99%

Amino acids 149 and 294 of human lecithin:cholesterol acyltransferase affect fatty acyl specificity

Zhao

Gebre

Parks

2004

Journal of Lipid Research

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We identified two regions of human LCAT (hLCAT) that when mutated separately to the corresponding rat sequence (E149A and Y292H/W294F) and transiently expressed in COS-1 cells increased phospholipase A 2 (PLA 2 ) activity by 5.5-and 2.8-fold, respectively, and increased cholesteryl ester (CE) formation by 2.9-and 1.4-fold, respectively, relative to hLCAT using substrate particles containing 1-16:0,2-20:4-sn -glycero-3-phosphocholine (PAPC). In contrast, both activities with 1-16:0,2-18:1-sn -glycero-3-phosphocholine (POPC) substrate were similar among the three LCAT proteins. The triple mutant (E149A/Y292H/W294F) had increased PLA 2 activity with PAPC similar to that observed with the E149A mutation alone; however, unlike E149A, the triple mutant demonstrated a 50% decrease in activity with POPC for both PLA 2 activity and CE formation, suggesting an interaction between the two regions of LCAT. Additional mutagenesis studies demonstrated that W294F, but not Y292H, increased PLA 2 activity by 3-fold with PAPC without affecting activity with POPC. The E149A/W294F double mutation mimicked the LCAT activity phenotype of the triple mutant (more activity with PAPC, less with POPC).In conclusion, separate mutation of two amino acids in hLCAT to the corresponding rat sequence increases activity with PAPC, whereas the combined mutations increase PAPC and decrease POPC activity, suggesting that these amino acids participate in the LCAT PC binding site and affect fatty acyl specificity. Lecithin:cholesterol acyltransferase (EC 2.3.1.43), a watersoluble glycoprotein secreted by the liver, is responsible for cholesteryl ester (CE) formation on the surface of plasma lipoproteins (1). The preferred lipoprotein substrate of LCAT is HDL, which contains the main activator of LCAT, apolipoprotein A-I (2). During the reaction, LCAT displays two enzymatic activities (3). The first is a phospholipase A 2 (PLA 2 ) step, in which the fatty acyl chain from the sn -2 position of phosphatidylcholine (PC) is cleaved to form an acyl-enzyme intermediate with the active site serine and the release of lyso-PC into solution. The subsequent step is an acyltransferase activity, in which the fatty acyl chain is transferred to the 3 ␤ -hydroxyl group of free cholesterol (FC) to generate CE (3). The CE product is extremely hydrophobic and partitions into the core of the HDL particle (4). LCAT plays a key role in the maturation of nascent HDL and in the reverse cholesterol transport pathway, a process in which excess FC in peripheral tissues is taken up by HDL particles, esterified by LCAT, and transported to the liver for uptake by the scavenger receptor class B type I (5).The fatty acyl composition of PC is the primary regulator of LCAT activity in plasma (1). It has been known for more than 30 years that human LCAT (hLCAT) prefers PC substrates containing 18:1 and 18:2 in the sn -2 position for CE synthesis, whereas rat and mouse LCAT prefer PC containing 20:4 in the sn -2 position (6-9). This fatty acyl preference was apparent even when ...

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Role of sn-2 Acyl Group of Phosphatidyl Choline in Determining the Positional Specificity of Lecithin-Cholesterol Acyltransferase

Cited by 42 publications

References 33 publications

Regulation of the Activity and Fatty Acid Specificity of Lecithin-Cholesterol Acyltransferase by Sphingomyelin and Its Metabolites, Ceramide and Ceramide Phosphate

Regulation of the Activity and Fatty Acid Specificity of Lecithin-Cholesterol Acyltransferase by Sphingomyelin and Its Metabolites, Ceramide and Ceramide Phosphate

Positional specificity of lysosomal phospholipase A2

Amino acids 149 and 294 of human lecithin:cholesterol acyltransferase affect fatty acyl specificity

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