©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vitro Expression of Structural Defects in the Lecithin-Cholesterol Acyltransferase Gene (*)

(Received for publication, January 25, 1995)

Hanns-Georg Klein (§) Nicolas Duverger (¶) John J. Albers (1) Santica Marcovina (1) H. Bryan Brewer Jr. Silvia Santamarina-Fojo (**)

From the Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 andthe Department of Medicine, Northwest Lipid Research Laboratories, University of Washington, School of Medicine, Seattle, Washington 98103

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Classic LCAT deficiency (CLD) and fish eye disease (FED) are two clinically distinct syndromes, associated with defects in the lecithin-cholesterol acyltransferase (LCAT) gene resulting in total (CLD) or partial (FED) enzyme deficiency. In order to investigate the underlying molecular mechanisms that lead to different phenotypic expression in CLD and FED, LCAT mutants associated with either CLD (LCAT, LCAT, and LCAT) or FED (LCAT, LCAT, LCAT, LCAT, LCAT, and LCAT) were expressed in vitro in human embryonic kidney 293 cells and characterized with respect to LCAT expression and enzyme activity. Evaluation of mutant LCAT gene transcription by Northern blot analysis demonstrated LCAT mRNA of normal size and concentration. Although all constructs gave rise to similar intracellular LCAT mass, the amount of enzyme present in the media for LCAT, LCAT, and LCAT was reduced to less than 10% of normal, suggesting that these mutations disrupted LCAT secretion. Western blot analysis of cell culture media containing wild type or mutant LCAT demonstrated the presence of a single normal-sized band of 67 kDa. The ability of the different enzymes to esterify free cholesterol in high density lipoprotein-like proteoliposomes (alpha-LCAT-specific activity) was reduced to less than 5% of normal for CLD mutants LCAT and LCAT and FED mutants LCAT, LCAT, LCAT, and LCAT, whereas that of LCAT, LCAT, and LCAT ranged from 45 to 110% of control. Although most FED mutant LCAT enzymes retained the ability to esterify free cholesterol present in alpha- and beta-lipoproteins of heat-inactivated plasma, esterification was undetectable in all CLD mutants (LCAT, LCAT, and LCAT). In contrast, all mutant enzymes retained the ability to hydrolyze the water soluble, short-chained fatty acid substrate p-nitrophenolbutyrate. In summary, our studies establish the functional significance of nine LCAT gene defects associated with either FED or CLD. Characterization of the expressed LCAT mutants identified multiple, overlapping functional abnormalities that include defects in secretion and/or disruption of enzymic activity. All nine LCAT mutants retained the ability to hydrolyze the water-soluble PNPB substrate, indicating intact hydrolytic function. Based on these studies we propose that mutations in LCAT residues 147, 156, 228 (CLD) and 10, 123, 158, 293, 300, and 347 (FED) do not disrupt the functional domain mediating LCAT phospholipase activity, but alter structural domains involved in lipid binding or transesterification.


INTRODUCTION

Lecithin-cholesterol acyltransferase (LCAT) (^1)is an important enzyme for extracellular cholesterol metabolism(1) . LCAT is secreted into the plasma by hepatocytes and catalyzes the conversion of free cholesterol into cholesteryl esters by transferring the second acyl chain from phosphatidylcholine to the 3-hydroxy group of cholesterol. Thus, LCAT plays a major role in the maturation of lipoprotein particles by providing cholesteryl esters which are incorporated into the core of plasma lipoproteins. In plasma, LCAT is preferentially associated with HDL (alpha-LCAT) and to a lesser extent with low density lipoprotein (beta-LCAT)(2) .

The mature LCAT polypeptide consists of 416 amino acids and has a calculated molecular mass of 47 kDa(3) . LCAT isolated from plasma has a molecular mass of 67 kDa due to post-translational processing involving N-linked glycosylation(4) . Although the tertiary structure of LCAT is not known, several important functional regions of the enzyme have been identified by chemical modification(5, 6, 7) , secondary structure analysis of the protein sequence(3, 8) , site-directed mutagenesis(9, 10, 11) , and DNA sequence analysis in patients with complete (CLD) or partial (FED) enzyme deficiency (12, 13, 14, 15, 16, 17, 18, 19, 20, 21) . These include the active site Ser, which is part of the serine esterase consensus sequence, the free cysteine residues 31 and 184, an alpha-helical segment extending from Glu to Lys, as well as the potential N-linked glycosylation sites at residues 20, 84, 272, and 384. Interestingly, structural LCAT gene defects in subjects with primary LCAT deficiency syndromes do not cluster around a particular area but involve all regions of the LCAT gene and result in different phenotypic expression of LCAT deficiency. The mechanisms by which these defects lead to decreased plasma LCAT activity are not known.

In this article we investigate functional significance of molecular defects in the LCAT gene that result in the expression of two phenotypically distinct syndromes, CLD and FED. Evaluation of nine different mutant LCAT enzymes identified various biochemical defects leading to either reduced or absent LCAT activity. The catalytic domain involved in mediating the phospholipase activity of the enzyme, however, was not affected, indicating that these mutations disrupted other domains in the LCAT structure. Our studies indicate that the different functional abnormalities associated with LCAT gene defects are in part responsible for the heterogeneiy observed in primary LCAT deficiency syndromes.


MATERIALS AND METHODS

DNA Amplification by PCR

LCAT-specific 5`- and 3`-flanking oligonucleotide primers containing restriction sites for XbaI and HpaI, as well as primers containing specific mutations within the LCAT cDNA, were synthesized on a DNA synthesizer (Applied Biosystems, Inc., Foster City, CA, model 380B). The LCAT cDNA (a gift from Dr. J. McLean, San Francisco, CA) was amplified by PCR from a pUC19 clone(22) , which contained the coding region of the human LCAT gene as described (23, 24, 25) using an automated DNA Thermal Cycler (Perkin-Elmer Cetus) and Pfu DNA polymerase (Stratagene, La Jolla, CA). Forty cycles were performed, each consisting of 45-s denaturation at 95 °C, 2-min annealing at 50 °C, and 3-min extension at 72 °C.

LCAT cDNA Expression Vector

The strategy for construction of the LCAT cDNA expression vector has been previously reported(20) . Briefly, restriction sites for XbaI and HpaI were introduced into the human LCAT cDNA by PCR amplification. The LCAT cDNA was ligated into the XbaI and HpaI sites of a pUC18 vector(26) , containing the cytomegalovirus immediate early promoter and the SV40 polyadenylation signal. Clones carrying the LCAT cDNA insert were identified by PCR using cytomegalovirus and SV40-specific primers and grown overnight at 37 °C in LB broth (Biofluids, Rockville, MD). DNA was isolated by minipreparation and the LCAT cDNA sequence verified by sequence analysis as described(20) . The recombinant vectors were amplified and purified by the cesium chloride double-banding method(27) .

Site-directed Mutagenesis of the LCAT cDNA

Specific mutations, known to be associated with complete or partial enzyme deficiency (Fig. 1), were introduced into the LCAT cDNA by the PCR overlap technique (28) using site-directed oligonucleotide primers (Fig. 1). Expression vectors carrying the mutant LCAT cDNA were generated as outlined above.


Figure 1: Structure of the human LCAT genomic and cDNA, location and of mutant residues (upper panel), and mutant codons associated with partial or classic LCAT deficiency (lower panel).



Transient Expression of the LCAT cDNA in Human Embryonic Kidney-293 Cells

Forty µg of the control expression vector without the LCAT insert or the recombinant plasmids containing wild type or mutant LCAT cDNA were transfected into subconfluent monolayers of kidney-293 cells using the calcium phosphate coprecipitation method (29) . All transfections were carried out in triplicate. The cell culture media (Dulbecco's modified Eagle's medium, Life Technologies, Inc.) were replaced with fresh media after 16 h and harvested after 48 h. Intracellular proteins were extracted by sonication of the pellet from collected cells as described(20) . Aliquots of the media and the intracellular extracts were kept at -70 °C until LCAT assays were performed.

Extraction of LCAT mRNA and Northern Blot Analysis

Total RNA was extracted from harvested cells using the guanidinium thiocyanate procedure(30) . Following separation on a 6% formaldehyde, 1% agarose gel, the RNA was transferred to Nytran (Schleicher & Schuell) and linked to the membrane using the UV Stratalinker (Stratagene, La Jolla, CA). A full-length LCAT cDNA probe was prepared from the LCAT expression vector by double digestion with XbaI and HpaI and labeled with [alpha-P]dCTP using a random primer labeling kit (DuPont NEN). The membrane was hybridized overnight at 42 °C by adding 20 times 10^6 disintegrations/min of the probe to the prehybridization solution. The filter was subsequently washed 4 times 15 min in 2 times SSC, 0.1% SDS at room temperature and 2 times 30 min in 0.1 times SSC, 0.1% SDS at 42 °C and the radioactivity quantitated on a Betagen (Betagen, Waltham, MA). The membrane was then stripped and rehybridized with [alpha-P]beta-actin for normalization of the LCAT mRNA data.

LCAT Mass

LCAT mass was quantitated by radioimmunoassay using a polyclonal antibody and I-human LCAT as described previously in detail(31) .

Western Blot Analysis of Culture Media from Transfected Cells

Ten ng of normal or mutant LCAT and molecular weight standards were loaded on a 4-20% denaturing, reducing sodium dodecyl sulfate-polyacrylamide gel and separated at 60 mV for 2 h. Protein bands were transferred onto a nitrocellulose membrane (Schleicher & Schuell) and visualized using a LCAT-specific peroxidase-coupled polyclonal antibody.

Cholesterol Esterification Rate (CER) and alpha-LCAT Activity

The CER was quantitated by measuring the rate of esterification of [^14C]cholesterol using autologous plasma as a substrate as previously reported(32, 33) .

The HDL-associated alpha-LCAT activity in plasma and transfection media was determined using an artificial HDL-like proteoliposome substrate as described previously(34) . Stable proteoliposomes were synthesized by 30 min of incubation of apoA-I, [^14C]cholesterol, and egg phosphatidylcholine at a molar ratio of 0.8:12.5:250 at 37 °C, and the alpha-LCAT activity was determined from the rate of formation of [^14C]cholesteryl ester.

Hydrolytic LCAT Activity against p-Nitrophenolbutyrate (PNPB)

To evaluate the effects of the structural LCAT gene defects on the phospholipase activity, the activity of in vitro expressed normal and mutant LCAT was tested using the water-soluble PNPB, which has previously been shown to be a substrate for LCAT(35) . Ten µl of a freshly prepared 50 mM PNPB stock solution was added to 1 ml of serum-free transfection media, and the release of p-nitrophenoxide was followed for 90 min at 28 °C in a UV spectrophotometer (Gilford Response, Ciba-Corning, Medfield, MA) at 400 nm. The hydrolytic LCAT activity was calculated for the linear range (0-60 min) of the reaction after subtraction of background values. The slope factor for each absorbance plot was determined from eight subsequent measurements using the computer program STATPLAN (The Futures Group, Glastonbury, CT) and the expression v = m times t + b. Molar hydrolytic rates were calculated using the term,

in which E is the molar extinction coefficient for a pH of 7.4 (1.55 times 10^4M cm) as previously reported (35) , and m the calculated slope factor for the absorbance plot.


RESULTS

LCAT Gene Mutants

Fig. 1illustrates the defects in the LCAT gene either associated with partial (FED) or classic LCAT deficiency (CLD) which were analyzed by in vitro expression.

Northern Blot Analysis of LCAT mRNA

Cells transfected with normal or mutant LCAT cDNA synthesized LCAT mRNA that was similar in size to that of normal LCAT mRNA (1.55 kilobases) as demonstrated by Northern blot hybridization (Fig. 2). Densitometric analysis of the hybridization signals using the LCAT and beta-actin probes revealed normal LCAT mRNA levels in cells transfected with mutant constructs when compared to normal (data not shown).


Figure 2: Northern blot analysis of cellular extracts of kidney-293 cells transfected with normal or mutant LCAT cDNA. Normal size LCAT mRNA is visualized by autoradiography following hybridization with a full-length LCAT cDNA probe. The beta-actin standard is shown below. NL, normal.



LCAT Mass

All constructs gave rise to similar intracellular LCAT concentrations (0.3 ± 0.1 to 0.8 ± 0.4 µg); however, the amount of mutant LCAT secreted into the culture media ranged from zero (LCAT) to 134% (LCAT) of control (Table 1). Thus, it appears that substitution of residues 147 and 156 as well as deletion of residue 300 reduces secretion of LCAT to less than 7% of control.



Western Blot Analysis of Culture Media from Transfected Cells

Immunoblotting of transfection media containing normal or mutant LCAT demonstrated a single band at 67 kDa (Fig. 3), corresponding to the fully glycosylated LCAT present in plasma(4) . Mutant LCAT was not detected in the media.


Figure 3: Western blot analysis of cell culture media transfected with normal and mutant LCAT cDNA. Equal amounts of transfection media were loaded on a denaturing, reducing SDS-gel, transferred onto a nitrocellulose membrane, and stained with an specific peroxidase-coupled LCAT-antibody. A single band of 67 kDa was detected in the transfection media of all constructs. A semiquantitative correlation was found compared to data from the radioimmunologic LCAT quantitation (Table 1). NL, normal.



CER and alpha-LCAT Activity

CER, a measure of cholesterol esterification in both alpha- and beta-lipoproteins, was assayed using heat-inactivated plasma(33) , while the HDL-like proteoliposome substrate was used to quantitate alpha-LCAT activity (Fig. 4). As expected from the plasma studies, the CLD mutants (LCAT, LCAT, and LCAT) had virtually no detectable CER using the heat-inactivated plasma substrate, whereas the ability of the FED mutants (LCAT, LCAT, LCAT, LCAT, LCAT, and LCAT) to esterify free cholesterol in plasma (CER) was highly variable, ranging from zero to 170% of control. Using the proteoliposome substrate, a wide range of specific activities were detected in both CLD and FED mutants, ranging from zero (LCAT) to 115% of control (LCAT). With the exception of the CER which was absent in all three CLD mutants, our studies failed to identify a consistent difference in the ability of CLD versus FED mutants to utilize the two different substrates. In fact, the functional properties of expressed FED mutant LCAT were undistinguishable from that of CLD mutant LCAT. On the other hand, the specific CER and alpha-LCAT-specific activity for LCAT was greater than 100% that of normal LCAT. Thus, it appears that partial LCAT deficiency associated with this mutation is a result of reduced secretion of a fully functional LCAT ( Fig. 4and Table 1).


Figure 4: Specific enzymic activities of mutant LCAT for three different substrates: Heat-inactivated control plasma (CER), HDL-like proteoliposomes (alpha-LCAT activity), and PNPB (hydrolytic phospholipase activity). Data shown represent means ± S.D. from duplicate measurements of triplicate transfections. NL, normal.



Hydrolytic LCAT Activity against PNPB

In contrast to the plasma and the proteoliposome substrates, which require interaction of the enzyme with the lipid-soluble interphase of the substrate, the specific hydrolytic activities of mutant LCAT against the water-soluble substrate PNPB were not significantly different from the control (Fig. 4, bottom panel), indicating that the phospholipase activity of the enzyme was not disrupted by the studied amino acid substitutions.

Secondary Structure Analysis

Computer-aided secondary structure analysis (PC/GENE, Intelli-Genetics, Mountain View, CA) using three different prediction methods (36, 37, 38) revealed no major rearrangements of the predicted secondary structure of the mutant LCAT enzymes. Analysis of the predicted alpha-helical segment extending from residues 154 to 167 with the program HELWHEEL (PC/GENE) revealed a hydrophobic moment of 0.52 for the wild type protein, suggesting that this segment has amphipathic properties. A reduction of the hydrophobic moment to 0.47 for LCAT(TyrAsn) and to 0.35 for LCAT(ArgCys) was found for the two mutations affecting this region. The replacement of Tyr by Asn is not within 20 °C of the center of the nonpolar face, and therefore may not be a helix braker(39) , whereas substitution of Arg by Cys is within 40 °C from the center of the hydrophilic face of the alpha-helical segment and probably leads to termination of the helical conformation(39) .


DISCUSSION

In the past several years, a number of molecular defects in the LCAT gene that result in the expression of two clinically distinct syndromes, CLD and FED, have been identified in selected kindreds using DNA sequence analysis(12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 40, 41) . The underlying mechanisms, however, which lead to the loss of enzyme activity in CLD and FED, are not understood. In fact, the functional significance of most of these LCAT gene defects has not yet been established.

In the present study we establish the functional abnormalities associated with nine naturally occurring structural defects in the LCAT gene by in vitro expression of normal and mutant LCAT in human embryonal kidney-293 cells. The concentration and the specific HDL-associated alpha-LCAT activity achieved in the transfection media using this expression system is equivalent to the normal human LCAT plasma levels(20, 31, 34) , resulting in significantly higher LCAT concentration and activity than previously described in other expression systems(9, 12, 42) . In addition, the specific HDL-associated alpha-LCAT activity of the enzyme in the culture media of human embryonic kidney-293 cells transfected with the normal LCAT cDNA was similar to those reported for plasma LCAT(20, 31, 34) .

The levels of expression achieved by the different LCAT mutants in our transfection system were similar to those observed by plasma analysis of the individual LCAT-deficient subjects. Thus, the LCAT mass of in vitro expressed mutant LCAT was either similar to control (AsnLys, ThrIle, ProLeu), moderately reduced (MetIle, ArgCys, ThrMet) or decreased to less than 7% of control TyrAsn, LCAT, ArgTrp). The LCAT mutations analyzed did not appear to disrupt transcription or translation, as demonstrated by normal levels of LCAT mRNA and intracellular LCAT mass; however, low LCAT levels present in the cell culture media of mutant LCAT, LCAT, and LCAT suggest that residues 147, 156, and 300 may result in disruption of normal LCAT secretion or lead to intracellular degradation of the mutant enzyme.

In order to analyze the ability of the mutant LCAT enzymes to utilize different substrates, the specific LCAT activities using either heat-inactivated control plasma (CER), HDL-like proteoliposomes (alpha-LCAT activity), or the water-soluble PNPB were evaluated. The CER measures cholesterol esterification in both, alpha- and beta-migrating lipoproteins. Using the plasma substrate, LCAT activity was detectable only in those mutants, associated with partial LCAT deficiency and the FED phenotype. Consistent with the findings in the plasma of CLD patients, LCAT, LCAT, and LCAT had no detectable CER. Calculation of the specific CER revealed similar to normal activities for FED mutants LCAT and LCAT, whereas FED mutants involving residues 10, 123, 293, and 347 had less than 10% of control specific activity against the plasma substrate.

The HDL-associated alpha-LCAT activity was quantitated using synthetic HDL-like proteoliposomes. This assay selectively reflects cholesterol esterification in alpha-lipoproteins, yet it requires like the CER assay interaction of the enzyme with its substrate in a lipid-aqueous interphase. Our findings indicate, that, despite original proposals (43, 44) suggesting that CLD and FED can be distinguished biochemically by total (alpha- and beta-LCAT) or selective (alpha-LCAT) loss of enzyme activity, specific alpha activity, ranging from 10 to 110% of control was present in the culture media of most mutants.

Interestingly, two defects involving residues 156 and 158, which are located near Arg, are part of a predicted alpha-helix with amphipathic properties(8, 20) . This alpha-helical segment, extending from Glu to Lys has been proposed to function as an interfacial binding site for LCAT with its lipid substrates. Although LCAT and LCAT result in reduction of the hydrophobic moment and eventually disrupt the alpha-helix, the residual specific alpha activities were 30-50%. This segment may therefore not be essential for interaction with the lipid substrate as previously suggested(8) , but may instead confer properties of substrate specificity.

The enzymic activity of all nine LCAT mutants was then analyzed using the water-soluble, short chain fatty acid substrate PNPB. The suitability of this substrate to measure the phospholipase activity of the LCAT reaction has been previously established(35) . Hydrolysis of this substrate occurs in the absence of a lipid interphase and thus does not require an intact lipid-binding domain. In addition, hydrolysis of PNPB reflects the initial phospholipase function of LCAT independent of transesterification since following hydrolysis the acyl chain is released into the buffer. In contrast to the variable residual activities of the mutant LCAT enzymes obtained with the proteliposome or the plasma substrate, the specific LCAT activities of all mutants for PNPB were greater than 50% that of control, suggesting that the phospholipase activity of the mutant enzymes was minimally affected. Our data therefore suggest that the structural domain involving the hydrolytic function of the enzyme is not disrupted by the amino acid substitutions introduced by the LCAT mutations analyzed. Instead, the loss of activity when either plasma or proteoliposomes were used as substrates may result from a conformational change of the LCAT polypeptide which affects the functional domain involved in binding to the lipid substrate, acyl transfer to the acceptor cholesterol, or the substrate specificity of the enzyme.

In summary, the functional significance of nine naturally occurring LCAT mutations phenotypically associated with complete or partial enzyme deficiency has been established. The functional explanations for partial enzyme deficiency include 1) reduced secretion of a fully functional enzyme (Leu), 2) reduced secretion of a partially active enzyme with variably reduced activity (ArgCys, TyrAsn, ThrMet, MetIle), and 3) normal secretion of an enzyme with reduced activity (ProLeu, ThrIle). Our data indicate that despite the loss of activity of most LCAT mutants against its natural substrates, all LCAT mutants retained their phospholipase activity, indicating that the functional domain involved in hydrolysis of the acyl chain is not affected by the mutation. Thus, the loss of esterification activity observed in these mutants may reflect conformational changes resulting in the disruption of the functional domains mediating either the transesterification or the lipid binding properties of the mutant LCAT enzymes.


FOOTNOTES

*
This study was supported in part by National Institutes of Health Grant HL 3-00H6 (to J. J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institute of Clinical Chemistry, Klinikum Grosshadern, University of München, Munich 15, Germany.

Recipient of a Postdoctoral Fellowship from Rhone-Poulenc-Rorer, Vitry sur Seine, France.

**
To whom correspondence should be addressed: National Institutes of Health, NHLBI, Molecular Disease Branch, Bldg. 10, Rm. 7N115, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-402-0521; Fax: 310-402-0190.

(^1)
The abbreviations used are: LCAT, lecithin-cholesterol acyltransferase; apo, apolipoprotein; CLD, classic LCAT deficiency; FED, fish eye disease; HDL, high density lipoproteins; CER, cholesterol esterification rate using plasma substrate; PCR polymerase chain reaction; PNPB, p-nitrophenolbutyrate.


ACKNOWLEDGEMENTS

We greatly appreciate the excellent technical assistance of Glenda Talley and Marie Kindt.


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