(Received for publication, January 25, 1995)
From the
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 (
-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
- and
-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.
Lecithin-cholesterol acyltransferase (LCAT) ()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 (
-LCAT) and to a lesser extent
with low density lipoprotein (
-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
-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.
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).
The HDL-associated -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, [
C]cholesterol, and
egg phosphatidylcholine at a molar ratio of 0.8:12.5:250 at 37 °C,
and the
-LCAT activity was determined from the rate of formation
of [
C]cholesteryl ester.
in which E is the molar
extinction coefficient for a pH of 7.4 (1.55
10
M
cm
) as
previously reported (35) , and m the calculated slope
factor for the absorbance plot.
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 -actin
standard is shown below. NL,
normal.
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.
Figure 4:
Specific enzymic activities of mutant LCAT
for three different substrates: Heat-inactivated control plasma (CER),
HDL-like proteoliposomes (-LCAT activity), and PNPB (hydrolytic
phospholipase activity). Data shown represent means ± S.D. from
duplicate measurements of triplicate transfections. NL,
normal.
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 -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
-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, Thr
Ile,
Pro
Leu), moderately reduced
(Met
Ile, Arg
Cys,
Thr
Met) or decreased to less than 7% of control
Tyr
Asn, LCAT
,
Arg
Trp). 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
(-LCAT activity), or the water-soluble PNPB were evaluated. The
CER measures cholesterol esterification in both,
- and
-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 -LCAT activity was quantitated using synthetic
HDL-like proteoliposomes. This assay selectively reflects cholesterol
esterification in
-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 (
- and
-LCAT) or
selective (
-LCAT) loss of enzyme activity, specific
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
-helix with amphipathic
properties(8, 20) . This
-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
-helix, the residual specific
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 (Arg
Cys,
Tyr
Asn, Thr
Met,
Met
Ile), and 3) normal secretion of an enzyme with
reduced activity (Pro
Leu,
Thr
Ile). 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.