(Received for publication, May 11, 1995; and in revised form, July 28, 1995)
From the
Heterozygosity for a 5-kilobase (kb) deletion of the first two
ligand-binding repeats (exons 2 and 3) of the low density lipoprotein
(LDL) receptor (R) gene (LDL-R 5kb) confers familial
hypercholesterolemia (FH). The FH phenotype is unexpected based on
previous site-directed mutagenesis showing that deletion of exons 2 and
3 resulted in little or no defect in LDL-R activity. In the present
study, we took unique advantage of the ability to distinguish the LDL-R
5kb from the normal receptor on the basis of size, in order to
resolve this apparent discrepancy. Fibroblasts from heterozygotes for
the LDL-R
5kb displayed 50% of normal capacity to bind LDL and
-VLDL, apparently due to lower receptor number. Cellular mRNA for
the
5kb allele was at least as abundant as that for the normal
allele. Immunoblotting and cell binding assays with anti-LDL-R antibody
IgG-4A4 demonstrated normal synthesis and transport of the
5kb
receptor. Ligand blotting demonstrated that the
5kb receptor
displayed minimal or no ability to bind LDL or
-VLDL. Thus, in
contrast to transfected cell lines, in human fibroblasts, the first two
cysteine-rich repeats of the LDL-R appear functionally necessary. These
characteristics of the LDL-R
5kb in human fibroblasts explain the in vivo phenotype of carriers.
The low density lipoprotein (LDL) ()receptor (R)
binds and catabolizes apolipoprotein E-containing chylomicron and VLDL
remnants and LDL. In the liver, LDL-R functions to remove these
lipoproteins from plasma for eventual excretion of the cholesterol into
the bile. In peripheral cells, it functions to provide the cell with
cholesterol needed for membrane synthesis. The LDL-R contains five
major structural domains: a seven-repeat, cysteine-rich ligand binding
domain encoded by exons 2-6, an epidermal growth factor-precursor
homology domain (exons 7-14), a glycosylation domain (exon 15), a
membrane-spanning domain (exon 16), and a cytoplasmic tail (exon
17)(1) . The LDL-R is one of the few proteins for which
knowledge of the structure-function relationship has been generated
both from site-directed mutagenesis and from numerous naturally
occurring human mutations.
Mutations in the LDL-R gene resulting in
a dysfunctional receptor cause a codominantly inherited disorder of
plasma cholesterol catabolism known as familial hypercholesterolemia
(FH). Human LDL-R mutations have been assigned to five classes of
defects based on their phenotypic effects on the receptor
protein(1) . We have previously described a deletion of
approximately 5 kb, which removes exons 2 and 3 of the LDL-R gene
(LDL-R 5kb)(2) . In site-directed mutagenesis experiments,
deletion of the first repeat (exon 2) has no effect on the binding or
internalization of LDL or
-VLDL or recycling of receptors in
transfected mammalian cells(3) . Simultaneous deletion of exons
2 and 3 has resulted in a receptor which binds LDL 70% as well as the
normal receptor and which binds
-VLDL equally as well(4) .
These results have led to the suggestion that the first two repeats of
the LDL-R ligand-binding domain are not necessary for LDL-R function.
Some studies have shown that the clinical phenotype resulting from
LDL-R mutations correlates with biochemical phenotype or
class(5, 6) . As such, one would expect that
heterozygosity for the LDL-R 5kb would result in relatively mild
or no expression of familial hypercholesterolemia (FH). However, taking
advantage of genetic founder effects among French Canadians, we have
observed that plasma total and LDL cholesterol levels among 8 probands
for this deletion are indistinguishable from those in heterozygotes for
a null LDL-R allele. In the context of a clinical genetic study of a
kindred with the 5-kb LDL-R gene deletion(7) , we noted that
heterozygote (HTZ) fibroblasts displayed consistently 50-60% the
maximal receptor activity of normal cells. This was again unexpected
based on the apparent activity of the LDL-R lacking exons 2 and 3 in
transfected cells.
Normally, the biochemical consequences of LDL-R
mutations in cells from carriers are difficult to study in the absence
of a homozygote. Unlike the case for the vast majority of described
mutations of the LDL-R, the LDL-R 5kb deleted protein is
distinguishable from the normal receptor on the basis of size. This
situation offers a unique opportunity to examine and compare the mRNA,
protein, and ligand binding to the mutant receptor with those of the
normal receptor in vivo within the same cell. The present
study provides evidence for important differences in the consequences
of deletion of exons 2 and 3 from the LDL-R gene as assessed by
site-directed mutagenesis and by analysis of heterozygous fibroblasts.
Binding
experiments with I-LDL at 4 °C and Scatchard analysis (Fig. 1A) revealed that the apparent defect in LDL-R
activity in LDL-R
5kb fibroblasts was due to an apparently lower
number of binding sites (94 versus 159 ng of ligand/mg of
cellular protein for LDL-R
5kb HTZ and normal fibroblasts,
respectively) with no difference in receptor affinity (2.96 and 3.12
µg/ml) compared to normal subjects. A similar experiment with
-VLDL as ligand revealed a defect in binding by
5kb
fibroblasts of approximately 50% that was also associated with lower
receptor number (205 versus 457 ng/mg) and with higher
affinity (0.42 versus 0.83 µg/ml) (Fig. 1B).
Figure 1:
Surface
binding of I-LDL and
I-
-VLDL to normal
and LDL-R
5kb HTZ fibroblasts. After incubation for 48 h in 10%
LPDS, fibroblasts were incubated at 4 °C with 1 ml of medium
containing the indicated concentrations of
I-LDL in the
presence or absence of a 20-fold excess of unlabeled LDL (A)
or
I-
-VLDL in the presence or absence of a 50-fold
excess of unlabeled
-VLDL (B). After 3 h, the total
radioactivity bound to cells was determined as described under
``Materials and Methods.'' Values shown are corrected for
nonspecific binding and represent the means of duplicate wells.
Nonspecific binding averaged 10% of total. Inset shows
Scatchard analysis.
Figure 2:
LDL-R mRNA from LDL-R 5kb and normal
allele. Reverse transcription-polymerase chain reaction of total RNA
extracted from LDL-R
5kb HTZ or normal fibroblasts was performed
with primers in exons 1 and 4 of the LDL-R gene. Shown is the output
from the automated Sequencer; peak areas are proportional to
fluorescence intensity. The peaks of 441 and 638 base pairs represent
amplified cDNA from the LDL-R
5kb and normal allele, respectively. FCS, fetal calf serum.
Figure 3:
Immunodetection of LDL-R in protein
extracts from normal and LDL-R 5kb HTZ fibroblasts. Forty µg
of fibroblast protein extracts were applied to 6% SDS-polyacrylamide
gels. Membranes were probed with IgG-C7 which recognizes the first
repeat of the ligand-binding domain (upper left) or with
IgG-4A4 directed against the carboxyl-terminal 14 amino acids of the
LDL-R (3) (lower left). Shown are the autoradiograms.
The source of fibroblasts is indicated between panels:
10Kb
HMZ, receptor-negative fibroblasts from a homozygote for the
French Canadian >10-kb LDL-R deletion(8) ;
5Kb, from LDL-R
5kb HTZ fibroblasts; N,
normal fibroblasts. Molecular mass in (kDa) is indicated at right; bands at 140 and 127 represent the normal and
5kb
form of the LDL-R, respectively. Right panel shows the results
calculated from densitometric scans of autoradiograms. Bars representing LDL-R
5kb HTZ fibroblasts are averaged band
intensities relative to the normal form of 3-4 independent
experiments with fibroblasts from 4 LDL-R
5kb HTZ and 2 normal
subjects.
The
relative amounts of LDL-R protein was assessed by densitometry in 4
independent experiments. The amount of total LDL-R protein was similar
in normal and 5kb HTZ fibroblasts (Fig. 3, lower
right). The amount of protein corresponding to the normal and
LDL-R
5kb allele was approximately equal in fibroblasts from 4
LDL-R
5kb HTZ (Fig. 3, left, LDL-R IgG-4A4, and lower right panel). No LDL-R protein was detected with
extracts from receptor-negative fibroblasts with either antibody (Fig. 3, left,
10kb HMZ). Thus, the LDL-R
5kb
protein appears to be synthesized in normal amounts.
Treatment of
cell protein extracts from LDL-R 5kb HTZ fibroblasts with
neuraminidase reduced the apparent size of the normal receptor from 147
to 134 kDa (Fig. 4), consistent with previous reports of
10-15-kDa reduction(19, 20) . A reduction of
apparent molecular mass was also observed for the
5kb protein,
from 134 to 117 kDa. The difference in size between the normal and
deleted receptor was similar before and after neuraminidase treatment.
Thus, no defect in glycosylation of the
5kb receptor was detected.
Figure 4:
Effect of neuraminidase on the molecular
mass of LDL-R of LDL-R 5kb (
5Kb) and normal (N) fibroblasts. Forty µg of fibroblast protein extracts
were treated with or without 0.016 unit of neuraminidase and subjected
to immunoblotting with IgG-4A4 (see ``Materials and
Methods''). Molecular mass (kDa) is indicated at right,
bands at 147 and 134 represent the normal LDL-R nontreated and treated,
respectively; bands at 134 and 117 represent the LDL-R
5kb
nontreated and treated, respectively.
Figure 5:
Binding of anti LDL-R antibody IgG- 4A4 to
cell surface of normal and LDL-R 5kb HTZ fibroblasts. Surface
binding at of
I-labeled IgG-4A4 to normal (NL, triangles) and LDL-R
5kb HTZ (
5Kb, circles) fibroblasts. After a 48-h incubation with 10% LPDS,
cells were incubated with 1 ml of medium containing the indicated
concentrations of
I-IgG-4A4 (485 cpm/ng). After 3 h at 4
°C, the total radioactivity bound to cells was determined as
described under ``Materials and Methods.'' Shown is specific
binding, obtained by subtraction of binding in the presence of a
20-fold excess of unlabeled antibody from the total. Nonspecific
binding averaged 40-50% of total. Due to anomalously high
nonspecific binding to NL fibroblasts at 1 µg/ml, this point was
not included in the curve. Inset shows Scatchard
analysis.
Figure 6:
Ligand blotting with I-LDL
to solubilized LDL receptors from normal and LDL-R
5kb HTZ
fibroblasts. Ninety µg of detergent-solubilized fibroblast protein
extracts were electrophoresed on 6% SDS-polyacrylamide gels. After
transfer to nitrocellulose, membranes were probed with 17 µg/ml
I-LDL (100-300 cpm/ng) (top) or 35
µg/ml
I-
-VLDL (80 cpm/ng) (bottom).
Shown are autoradiograms. Molecular mass (kDa) is indicated at the right; bands at 140 and 127 represent the normal and
5kb
form of the LDL-R, respectively. Top, extracts from
receptor-negative (lanes 1, 5, and 10),
normal (lanes 2 and 6), and LDL-R
5kb HTZ
fibroblasts (lanes 3, 4, and 7-9); bottom, from receptor-negative (lane 2), normal (lanes 3 and 4), and
5kb HTZ (lanes
5-10) fibroblasts.
Site-directed mutagenesis experiments have shown minimal loss
of LDL and -VLDL binding, respectively, from an LDL receptor
lacking exons 2 and 3 encoding the first two of seven ligand binding
repeats (3, 4) . Based on this information it is
surprising that in French Canadians, with the exception of carriers of
the
2 allele(7) , heterozygosity for a 5-kb deletion of
the LDL-R gene (LDL-R
5kb) encompassing exons 2 and 3 is
associated with plasma LDL and total cholesterol levels which are
equally as elevated as those associated with heterozygosity for an
LDL-R null allele. The present study sought to resolve this apparent
contradiction between in vivo phenotype and in vitro consequences of the LDL-R deletion.
Among possibilities to
explain the association of the LDL-R 5kb with FH were decreased
mRNA or protein synthesis, slow transport to the cell surface, or poor
affinity of the receptor for LDL. The first two possibilities were
ruled out by measurements of normal levels of mRNA and normal levels of
LDL-R protein in LDL-R
5kb HTZ fibroblasts. The receptor appeared
to be glycosylated normally, implying normal processing(21) .
LDL-R
5kb HTZ fibroblasts bound similar amounts of anti-LDL-R
antibody IgG-4A4 as did normal cells, also suggesting normal transport
of the
5kb receptor to the cell surface. However, ligand blotting
of cell protein extracts from LDL-R
5kb HTZ fibroblasts revealed
little or no LDL binding to the
5kb receptor. Thus, the apparent
reduction of 50% in receptor number in LDL-R
5kb HTZ fibroblasts
compared to normal is due not to the absence of the receptor on the
cell surface, such as in a class 2 receptor defect, but to the
inability of the receptor to bind LDL, i.e. a class 3 defect.
The deleted receptor was also defective in binding apolipoprotein E, as
evidenced by 50% of normal maximal cell surface binding of
-VLDL
in
5kb HTZ fibroblasts and by weak interaction with the deleted
receptor on ligand blots. This result is also in contrast to those
observed in the transfected receptor lacking exons 2 and 3, which
displayed no defect in
-VLDL uptake and to data which suggest that
LDL-R repeat 5 mediates binding of apolipoprotein E-containing
lipoproteins(22) .
Conversely to the present study, theoretically severe mutations in the LDL receptor gene do not always result in the FH phenotype. A class 2B defective LDL-R receptor (i.e. synthesized but not displayed on cell surface) was reported (23, 24) in which heterozygous parents of the affected homozygous child did not express consistent or significant hypercholesterolemia. An LDL-R gene deletion of approximately 10 kb, FH-Tonami-2(25) , eliminating exons 2 and 3, has been found in 10 Japanese families with hypercholesterolemia and is associated with cholesterol levels lower than those of typical FH patients, including two heterozygous family members with normal plasma cholesterol levels (26) . According to the present data, such cases of milder than expected FH phenotype are most likely explained by up-regulation of the normal allele in some cases rather than by residual function of the defective LDL-R.
Several possibilities could explain the discrepancy
between the consequences of deleting LDL-R exons 2 and 3 described
herein and those previously described(4) . The present
5-kb genomic deletion of the LDL-R gene between introns 1 and 3 (2) is predicted to result in an in-frame creation of an Ala
residue at the expense of Val
and Pro
; i.e. an amino-terminal sequence of
Ala
-Ala-Pro
. In the site-directed mutagenesis
study(4) , the deletion was of residues 1-83; i.e. an amino-terminal sequence of Pro
-Pro
.
The possible functional significance of this difference is not clear
but seems unlikely to account for differences in the ligand binding
ability of the two deleted receptors. Other examples of discrepancies
between apparent effects of gene deletions as assessed by in vitro studies and by phenotypic expression of a naturally occurring
deletion is seen when domain 3 (O-linked sugar domain) is
deleted in vitro by site-directed mutagenesis resulting in no
defect in receptor activity(27) , while a homozygote for such a
mutation expresses FH(28, 29) . A variant of
lipoprotein lipase containing an Asn291
Ser substitution which is
functionally mildly abnormal in vitro(30) is
associated with type IV hypertriglyceridemia in French
Canadians(31) . In the case of lipoprotein lipase, the
unexpectedly profound clinical effect of heterozygosity for a mildly
defective variant may be attributable to a dominant negative mechanism,
wherein the defective variant would interfere with lipoprotein lipase
dimerization, which is necessary for function. Although the LDL-R is
present on the cell surface as a monomer, one possible locus for a
dominant negative effect of heterozygosity for a defective receptor
could be in receptor clustering prior to
internalization(14, 32) .
However, such an
explanation for the defective LDL-R activity observed in LDL-R 5kb
HTZ fibroblasts is unlikely based on the observation that the
5kb
receptor is poorly able to bind ligand. A more likely explanation for
the discrepancies between results obtained from site-directed
mutagenesis studies and HTZ fibroblasts is the amount of receptor
expressed under each set of circumstances. Overexpression of proteins
in transfected cells has been observed to result in unphysiological
phenomena, such as secretion of immature forms of apolipoprotein A-I (33) or constitutive activity of sterol regulatory element
binding proteins 1 and 2(34) . Thus, prediction of in vivo phenotypic effects of gene mutations from their functional effects
in transfected cells may be complicated by the unphysiologically high
levels of expression.
It has been estimated that 15-30% of
``isolated'' O-linked carbohydrate is located on the
amino-terminal half of the receptor(27) , more specifically,
within the 40-kDa ligand-binding domain(35) . The absence
of glycosylation in this domain in a monensin-resistant cell line has
been shown to reduce LDL-R affinity for LDL by approximately
75%(35) . Thus, loss of O-linked carbohydrate may at
least partially explain the absence of ligand binding of the LDL-R
5kb receptor observed in the present study. Although similar
decreases in molecular mass after neuraminidase treatment between the
LDL-R
5kb and normal protein in the present study may imply that
significant O-linked glycosylation does not occur in the first
two repeats of the LDL-R, it is questionable whether a difference would
be detectable. O-Linked sialic acid and galactose residues are
expected to contribute approximately 25 kDa to the molecular mass of
the LDL-R (27) . Therefore, if the carbohydrate was evenly
distributed among repeats, the expected loss of molecular size after
neuraminidase treatment due to glycosylation of the first two repeats
is 3 kDa. Thus, it is possible that the effect of deletion of exons 2
and 3 on LDL-R activity is attributable to loss of carbohydrate and
subsequent loss of receptor affinity for LDL. As such, possible
differences in glycosylation patterns between human fibroblasts and
transfected CHO cells may contribute to differences between the present
and a previous study (4) on the functional consequence of the
absence of these two repeats.
Another potentially interesting
explanation for the surprisingly severe effects of the LDL-R 5kb
is a regulatory effect of a gene deletion that is not apparent in cells
transfected with cDNA. Thus, one possibility is that deletion of a
liver-specific enhancer in introns 1 or 2 causes a liver-specific
regulatory defect. An LDL-R gene deletion of exons 2 and 3 similar to
the LDL-R
5kb has been reported to result from Alu
recombination(36) . Alu sequences have been known to act as
enhancers (37) or repressors(37) . In the present
study, however, LDL-R mRNA corresponding to the
5kb allele was
consistently higher in fibroblasts and lymphocytes than that of the
normal allele, suggesting deletion of an element which may act as a
repressor, at least in these cell types. Further studies will explore
the regulatory consequences of the LDL-R
5kb.