(Received for publication, July 27, 1994; and in revised form, November 30, 1994)
From the
The well documented association between high plasma levels of
lipoprotein(a) (Lp(a)) and cardiovascular disease might be mediated by
the lysine binding of apolipoprotein(a) (apo(a)), the plasminogen-like,
multikringle glycoprotein in Lp(a). We employed a mutational analysis
to localize the lysine-binding domains within human apo(a). Recombinant
apo(a) (r-apo(a)) with 17 plasminogen kringle IV-like domains, one
plasminogen kringle V-like domain, and a protease domain or mutants
thereof were expressed in the human hepatocarcinoma cell line HepG2.
The lysine binding of plasma Lp(a) and r-apo(a) in the culture
supernatants of transfected HepG2 cells was analyzed by
lysine-Sepharose affinity chromatography. Wild type recombinant Lp(a)
(r-Lp(a)) revealed lysine binding in the range observed for human
plasma Lp(a). A single accessible lysine binding site in Lp(a) is
indicated by a complete loss of lysine binding observed for r-Lp(a)
species that contain either a truncated r-apo(a) lacking kringle IV-37,
kringle V, and the protease or a point-mutated r-apo(a) with a Trp-4174
Arg substitution in the putative lysine-binding pocket of
kringle IV-37. Evidence is also presented for additional lysine-binding
sites within kringles 32-36 of apo(a) that are masked in Lp(a) as
indicated by an increased lysine binding for the point mutant (Cys-4057
Ser), which is unable to assemble into particles. An important
role of these lysine-binding site(s) for Lp(a) assembly is suggested by
a decreased assembly efficiency for deletion mutants lacking either
kringle 32 or kringles 32-35.
Lipoprotein(a) (Lp(a)) ()is a cholesterol-rich
particle composed of a low density lipoprotein (LDL) core and a single
copy of the high molecular weight glycoprotein apolipoprotein(a)
(apo(a))(1) , covalently linked to apolipoprotein B-100 by a
single disulfide bridge(2, 3) . The physiological
function of Lp(a) is still unknown(1, 4) . There is
increasing evidence, however, that Lp(a) may be involved in
atherogenesis. Epidemiological studies have demonstrated a strong
association between high Lp(a) plasma concentrations and cardiovascular
disease(5, 6, 7) . Three recent prospective
studies (8, 9, 10) have reported conflicting
conclusions on the association of elevated Lp(a) levels and the risk of
coronary heart disease. In both the Lipid Research Clinics Coronary
Primary Prevention Trial (9) and the
Göttingen Risk, Incidence and Prevalence Study (10) elevated Lp(a) concentrations were an independent risk
factor for coronary heart disease in hypercholesterolemic white men.
The Physicians' Health Study(8) , however, found no
evidence for an association between Lp(a) levels and the risk for
future myocardial infarction. It should be noted that in this latter
study (8) the participants were on average 10 years older and
had lower mean cholesterol levels than those males recruited into the
other two studies(9, 10) . In atherosclerotic lesions
of coronary bypass patients Lp(a) has been shown to accumulate
proportionally to Lp(a) plasma levels(11, 12) .
Apo(a)transgenic mice expressing free apo(a) glycoprotein in their
plasma (13, 14) showed an increased susceptibility to
the development of atherosclerotic lesions when compared with normal
mice(13) . Occlusive arterial thrombosis has been observed in
cynomolgus monkeys with high Lp(a) plasma levels(15) . As yet,
the mechanisms by which Lp(a) might promote atherogenesis remain
obscure.
The discovery of a striking homology between apo(a) and
plasminogen (Pg) (16, 17) led to the hypothesis that
Lp(a) might interfere with fibrinolysis(18) . The fibrinolytic
proenzyme Pg consists of a preactivation peptide, five so-called
``kringle'' domains, numbered I-V, and a protease
domain (reviewed in (19) ). Several functions of Pg appear to
be enhanced by its kringle structures. Lysine-binding sites (LBSs)
within Pg kringles mediate the binding of Pg to
fibrin(ogen)(20, 21, 22) . Kringles I and IV
contain binding sites with moderate to high affinity for L-lysine and related -amino acid
analogs(23, 24, 25) , whereas a weak LBS has
been demonstrated in kringle V(26) . The three-dimensional
structure of Pg kringle IV and its complex with the lysine analog
-amino-caproic acid (EACA) has been determined by high resolution
x-ray crystallography (27, 28) revealing a preformed
lysine-binding pocket at the surface of the kringle structure. Kringle
IV of Pg has been shown to bind fibrinogen(29) . Apo(a)
contains 10 distinct kringle repeats arranged in tandem all with high
homology (61-75%) to kringle IV of Pg followed by single Pg-like
kringle V and protease domains(17) . As a result of an
extensive size polymorphism in the apo(a)
gene(1, 30) , more than 30 apo(a) isoforms have been
found in human plasma differing in the number of kringle IV repeats.
Based on their electrophoretic mobility apo(a) isoforms have been
classified as F, B, S1, S2, S3, and S4(31) . Recent in
vitro studies have revealed that Lp(a) may bind to and displace Pg
from its receptor at the surface of endothelial
cells(32, 33, 34) , monocytes (32, 35) and platelets(36) . Several
investigators have observed lysine binding (37, 38, 39) and fibrin(ogen)-binding (40, 41, 42) of Lp(a) and/or apo(a). The
latter is inhibited by EACA, indicating that LBSs in apo(a) are
involved in fibrin(ogen)-binding of Lp(a)(40, 41) .
Impaired Pg activation has been demonstrated in the presence of high
plasma levels of Lp(a)(40, 43) .
Knowledge of the structural basis for the lysine- and fibrin(ogen)-binding of Lp(a) is paramount to our understanding of the role of this lipoprotein in fibrinolysis. A sequence comparison between human and rhesus monkey Lp(a), which differ in their lysine binding abilities led Scanu et al.(44) to suggest that kringle 37 of apo(a) plays a dominant role in the binding of Lp(a) to lysine. Based on sequence comparisons and molecular modeling (45, 46) it has been predicted that not only kringle 37, containing the most highly conserved lysine-binding pocket relative to Pg kringle IV, but also kringles 32-35 comprise potential lysine-binding domains. Single apo(a) domains expressed in bacteria (47, 48) or in mammalian cells (49) revealed lysine binding activity in kringle 37 but not in kringles 1 and 2. Direct experimental evidence for the localization of LBSs in apo(a) and Lp(a) is still lacking.
We now describe a mutational analysis aimed at the identification of
lysine-binding domains in recombinant apo(a) (r-apo(a)).
Lysine-Sepharose affinity chromatography was employed to compare the
lysine binding activity of wild type and mutant r-apo(a)/r-Lp(a) in the
medium of transfected HepG2 hepatocarcinoma cells. In addition, we
performed immunoblotting experiments with wild type and mutant r-apo(a)
samples to assess a possible function of lysine-binding kringles in
apo(a) for the assembly of apo(a)apoB complexes.
Figure 1: Schematic representation of wild type (a) and mutant (b-i) apo(a) encoded by cDNA expression plasmids. Amino acid substitutions in the point mutants pCMV-A18_4174Arg and pCMV-A18_4057Ser are indicated under the corresponding kringles. The presence of a B in kringle 36 indicates the ability of apo(a) entities to form a disulfide bridge with apoB-100 in LDL. Oligonucleotides(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) and restriction sites involved in vector constructions are indicated: A, AvrII; B, BsmAI; C, Cfr10I; Cl, ClaI; E, EcoRV; Ea, EarI; N, NciI; S, SalI; Sm, SmaI.
Figure 2:
Immunoblot analysis of wild type and
mutant r-apo(a)s. Following transient transfection of HepG2 cells with
wild type and mutant apo(a) expression plasmids, culture supernatants
were analyzed by SDS-PAGE under nonreducing (panel A) or
reducing (panel B) conditions and subsequent immunoblotting. A
phenotyping standard (St) containing the apo(a) isoforms S1,
S2, S3, and S4 (31) has been included in panel B.
Samples contained the following r-apo(a)s: lane 1, A18 (wild
type); lane 2, 32-P; lane 3,
V-P; lane
4,
37-P; lane 5, Arg-4174; lane 6,
Ser-4057. The positions of high molecular weight apo(a)
apoB
complexes (c) and free apo(a) glycoproteins (A11 and A18) are indicated to the left of panel
A.
HepG2-derived wild type r-Lp(a) obtained after three independent transfections revealed lysine binding of 30.6 and 92.2%, respectively, when analyzed in the absence and in the presence of Tween (Fig. 3, panels A and G, Table 1). Binding of r-Lp(a) to the lysine-Sepharose matrix could be completely blocked by the addition of 50 mML-lysine to sample and washing buffer, an effect that was not seen by using L-glycine at the same concentration (data not shown). Additional evidence for the specificity of the employed lysine-Sepharose method came from the estimation of HepG2-derived albumin and apolipoprotein A-I in the chromatography fractions. In contrast to r-Lp(a), both proteins were detected exclusively in the fall-through but not in the EACA-eluted fractions (data not shown). SDS-PAGE/immunoblotting and densitometric scanning of the r-apo(a) bands were employed to confirm the ELISA quantification of Lp(a) in the chromatography fractions and to demonstrate the integrity of r-apo(a) in all fractions of the lysine-Sepharose chromatography (data not shown). Variation in the expression rate of r-apo(a) led to some interexperimental variation in the amount of loaded r-Lp(a), which did not affect the lysine-binding data as indicated by the standard deviations in Table 1.
Figure 3:
Elution profiles from the characterization
of r-Lp(a)/r-apo(a) by lysine-Sepharose affinity chromatography. Lysine
binding of r-Lp(a)/r-apo(a) contained in the medium of transfected
HepG2 cells was studied in the absence (panels A-F) and
in the presence of 1% Tween-20 (panels G-M). The Lp(a)
content of the fractions collected before and after the addition of
EACA was determined by ELISA. The following samples were analyzed: panels A and G, A18 (wild type); panels B and H, mutant V-P; panels C and I, mutant
37-P; panels D and K, mutant
32-P; panels E and L, mutant Arg-4174; panels F and M, mutant Ser-4057. The addition of EACA
to the elution buffer is indicated by an arrow.
Plasma samples from 10 healthy donors (see ``Experimental Procedures'') contained between 4 and 460 mg/liter Lp(a) without significant degradation of apo(a) isoforms as analyzed by immunoblotting (data not shown). Prior to affinity chromatography plasma samples were diluted 10-fold in HepG2-conditioned culture medium to mimic the binding conditions employed during the characterization of r-Lp(a). When the samples were analyzed in the absence of Tween, lysine binding expressed as percentage of Lp(a) bound to lysine-Sepharose (see ``Experimental Procedures'') varied between 5.2 and 55.8% (mean = 26.2%). The addition of Tween caused a strong increase in the lysine binding of each plasma Lp(a) sample to values between 40.8 and 94.6% (mean 69.8%). There was no correlation between lysine binding and the level of plasma Lp(a), nor was the lysine-binding ability correlated with the apo(a) isoform size (data not shown).
Sequence comparisons and molecular modeling (44, 46) have suggested that kringle 37 of Lp(a) has
retained the lysine binding properties observed in Pg kringle IV.
Therefore, we analyzed r-Lp(a) containing an apo(a) without kringle 37,
kringle V, and the protease domain for its lysine binding activity.
Following transfection of plasmid pCMV-A18 37-P (Fig. 1c) r-Lp(a) containing culture medium was
characterized by lysine-Sepharose chromatography. In the absence of
Tween, we detected only 3.7% lysine binding (Fig. 3, panel
C, and Table 1). When analyzed in the presence of Tween,
however, 68.6% of the
37-P mutant r-Lp(a) bound to
lysine-Sepharose (Fig. 3, panel I, and Table 1).
This value, intermediate between the binding of wild type (92.2%) and
32-P (0.9%) could be explained by the existence of additional LBSs
within kringles 32-36 of apo(a), which might be masked in Lp(a).
Direct evidence for this additional lysine binding activity independent
of kringle 37 was obtained by the characterization of the mutants
Ser-4057 and
36-P (Table 1). Because of a Cys
Ser
substitution of the single unpaired cysteine in kringle 36 the mutant
Ser-4057 apo(a) (Fig. 1g) is unable to form Lp(a)
particles (2) as seen from the analysis by nonreducing SDS-PAGE (Fig. 2A, lane 6). Analyzed in the absence of
Tween, this mutant revealed 53.4% lysine binding, which increased to
84.2% in the presence of Tween (Fig. 3, F and M, and Table 1). Mutant
36-P (Fig. 1d) lacking not only the ability to form Lp(a)
particles (data not shown) but also the kringle 37 LBS exhibited 35.2
and 96% lysine binding in the absence and in the presence of Tween,
respectively (Table 1).
Figure 4:
Immunoblot analysis of wild type and
mutant r-apo(a)s expressed by HepG2 cells. Culture supernatants from
transfected HepG2 cells were fractionated by SDS-PAGE under nonreducing
conditions followed by immunoblotting with apo(a) specific monoclonal
antibody 1A2. Lane 1, mutant Ser-4057; lane 2, apo(a)
phenotyping standard (Immuno); lane 3, A18 (wild type); lane 4, mutant 32; lane 5, mutant
32-35. The positions of free r-apo(a) glycoprotein (a) and the r-apo(a)
apoB complex (c) are
indicated to the right.
From the data presented here, we propose the existence of two
functionally distinct LBSs within the apo(a) glycoprotein (Fig. 5). LBS I is situated in kringle 37 of apo(a) and is
responsible for the 30.6% lysine binding of wild type r-Lp(a) particles
observed in the absence of Tween. This is concluded from a comparison
of the lysine binding activities determined for r-Lp(a)s containing
wild type apo(a) with r-Lp(a) containing either of the apo(a) mutants,
37-P (Fig. 1c) or Arg-4174 (Fig. 1f). The mutant apo(a)s retained the ability to
assemble into r-Lp(a) particles as shown by immunoblotting (Fig. 2A, lanes 4 and 5) and density
gradient centrifugation (data not shown). In contrast to wild type
Lp(a) both mutants failed to bind to the lysine matrix in the absence
of Tween. This result is in agreement with predictions based on
sequence comparisons and molecular
modeling(44, 45, 46) . The inability of
r-Lp(a) containing the Arg-4174 mutant apo(a) to bind to
lysine-Sepharose gives experimental support to the hypothesis (44) that the failure of rhesus monkey Lp(a) to bind to this
matrix is caused by the amino acid substitution Trp
Arg at the
position homologous to Trp-4174 in human apo(a). The deletion mutant
32-P did not bind to lysine-Sepharose either in the absence or in
the presence of Tween, indicating that the N-terminal apo(a) kringles
1, 2A, 30, and 31 do not contribute directly to the lysine binding
activity of Lp(a). In fact, these domains might interfere with the
overall lysine- and/or fibrin(ogen) activity of Lp(a) because of
intramolecular interactions with LBS I in kringle 37. Crystals of
tissue plasminogen activator kringle 2 represent an example where the
lysine-binding pocket of one kringle is occupied by an internal lysine
from another kringle(58) . Such intramolecular masking (Fig. 5) could explain both our finding of 30% lysine binding
activity for wild type r-Lp(a) and the significant interindividual
variation in lysine binding of plasma Lp(a) ranging from 5 to 55%. The
observation might result from differential masking by apo(a) isoforms
present in the plasma samples. This hypothesis is supported by a recent
report of apo(a) isoform-dependent fibrin(ogen) binding of plasma
Lp(a)s(59) . Clearly, further experiments are required to
evaluate the contribution of intra- and intermolecular masking of LBS I
within the Lp(a) particle.
Figure 5: Model for the arrangement of LBS I and II of apo(a) within r-Lp(a). LBS I (I), localized in kringle 37 of apo(a) is exposed in 30% of wild type r-Lp(a). Masking of LBS I by either intramolecular kringle-kringle interactions or intermolecular interactions of apo(a) with apoB-100 and/or other components in the culture medium might explain the failure of 70% of r-Lp(a) particles to bind to the lysine matrix as indicated in the right panel. LBS II (II) has been mapped to kringles 32-36. The number and exact localization of the kringles that form LBS II remains to be resolved. Within r-Lp(a) particles these site(s) are masked but can be exposed in the presence of Tween-20. Kringles 1-31 of r-apo(a), kringle V, and the protease domain do not contain detectable lysine binding activity(-).
We could also localize substantial lysine
binding activity to the region between kringles 32 and 35 (LBS II) in
apo(a). In contrast to LBS I, LBS II is only operationally defined and
may in fact represent lysine-binding sites within one or more of the
respective kringles. On the basis of molecular modeling, Guevara et
al.(46) proposed that in addition to kringle 37 the
kringles 32-35 but not kringle 36 also contain potential
lysine-binding sites. Further mutants will be required to accurately
map LBS II. LBS II is masked in the native r-Lp(a) particle since
neither the point mutant Arg-4174 nor the deletion mutant 37-P
demonstrated significant binding to the lysine affinity matrix in the
absence of Tween. In the presence of Tween 64.9% (Arg-4174) and 68.6%
(
37-P) lysine binding was observed with these two mutants.
Unmasking of LBS II in the presence of the detergent presumably
explains the increased lysine binding of both plasma Lp(a) and r-Lp(a)
species. Considering only wild type r-Lp(a) (Fig. 3, panels
A and G), the effect of Tween might be caused by an
increased accessibility of LBS I, e.g. by dispersal of
aggregates that render LBS I inaccessible. This cannot be the reason,
however, for our observations with the point and deletion mutants,
specifically with the 35.2% lysine binding for the deletion mutant
36-P in the absence of Tween, and a second LBS must be inferred.
We can only speculate about the molecular basis of the increase in
lysine binding of Lp(a) and r-Lp(a). Tween-sensitive intra- and/or
intermolecular interactions between apo(a) kringle domains might
normally mask LBS II. Alternatively or additionally, noncovalent
interactions between kringles 32-36 comprising LBS II and apoB in
LDL might cause the relatively low lysine binding of wild type apo(a)
(30.6%) and of the mutants
36-P (35.2%) and
V-P (33.6%) in
the absence of Tween, which increased to more than 90% in the presence
of the detergent. Proline-dependent interactions have been implicated
in the apo(a)
apoB complex (60) and might be one target
for the Tween effect. A Tween-sensitive association of apo(a) with
components in HepG2 conditioned culture medium might be another
explanation for the observed increase in lysine binding in the presence
of the detergent.
Because of a substitution of Cys-4057, which is
required for the assembly of r-Lp(a) (2) the expression of the
mutant Ser-4057 in HepG2 cells yields free apo(a) glycoprotein. A 53.4%
lysine binding of this mutant in the absence of Tween as compared with
30.6% for complexed wild type apo(a) suggests noncovalent interactions
between the LBS of apo(a) and lysine residues of apoB in LDL. As
hypothesized previously (2) , such interactions might be
involved in the positioning of cysteine residues in apo(a) and apoB
prior to disulfide bridging of the two proteins. This hypothesis is
supported but not proved by the decreased Lp(a) assembly observed for
the deletion mutants 32 and
32-35 (Fig. 4).
Additionally or alternatively to lysine binding, other noncovalent
interactions between the deleted domain(s) and LDL might be involved in
Lp(a) complex formation although the dose-dependent inhibition by EACA
of the in vitro association between transgenic apo(a) and
human LDL (14) argues in favor of a role of lysine binding
during Lp(a) assembly. An involvement of LBS I in the assembly of
r-Lp(a) can be excluded from our data since the mutants
37-P and
Arg-4174 were assembled into r-Lp(a) particles as effectively as wild
type apo(a).
Lysine-Sepharose affinity chromatography has been widely used to study in vitro the lysine binding characteristics of kringle proteins. Our mutational analysis provides an understanding of the structural elements determining the lysine binding properties of r-apo(a) and r-Lp(a). At this point, it should be stated that we cannot exclude the existence of additional low-affinity LBS in apo(a) that could not be detected with our model system because of the rather low concentrations of r-Lp(a)/r-apo(a) in the medium of transfected cells.
The substantial increase of lysine binding in the presence of Tween that we observed for both recombinant and plasma Lp(a) raises some questions about the physiological relevance of previous fibrin(ogen)-binding studies with plasma Lp(a)(41, 42) , which have been performed in the presence of Tween. Although the Tween concentrations in these studies were much lower than the one that we used in our lysine binding studies we were able to show that even the lowest concentration (0.01%), which has been used by Rouy et al.(41) , results in a 1.6-fold increase in lysine binding of wild type r-Lp(a) from 30 to 48% (data not shown). Tween might lead to the exposure of fibrin-binding sites that are not necessarily identical with the postulated LBS. The identification of lysine binding activity in the kringle 32-36 domains of apo(a) raises the possibility that the described fibrin binding of Lp(a) is (in part) secondary to the ``Tween effect.'' In the absence of a physiological ``Tween analog'' it may be important to re-evaluate the fibrin-binding activity of plasma Lp(a) without demasking LBS II by addition of Tween to the assay buffer.
Apart from the well documented size variation
of apo(a) isoforms in human plasma, recent data indicate a high degree
of conservation in the structure of apo(a)
isoforms(2, 61) , suggesting that our results obtained
with r-Lp(a) can be extrapolated to human plasma Lp(a). In human
atherosclerotic lesions Lp(a) has been co-localized with
fibrinogen(11, 62) . Co-localization of Lp(a) and
fibrinogen was not observed in cynomolgus macaques (15, 63) and in a hypercholesterolemic rhesus
monkey(64) , the latter probably being explained by the lack of
lysine binding described for rhesus monkey Lp(a)(44) . In order
to elaborate the hypothesis that lysine binding activity might
contribute to the atherogenicity and/or thrombogenicity of Lp(a) it
will now be important to use more physiological models to compare wild
type and mutant r-Lp(a) for its binding to cells and/or fibrin(ogen) in
the arterial wall. Without the availability of clinically defined Lp(a)
mutants differing in their lysine binding properties a comparison of
transgenic animals with circulating wild type or mutant Lp(a) seems to
be most appropiate to investigate the impact of lysine binding for the
proatherogenic and prothrombotic properties that have been attributed
to Lp(a) particles. However, the reported binding of r-apo(a) to the
arterial wall of apo(a) transgenic mice (13) and the resulting
interference with plasminogen activation (65) may be biased by
an unphysiological exposure of LBS II. The free transgenic apo(a)
glycoprotein circulating in these animals (14) has no
counterpart in human plasma, where apo(a) is found almost exclusively
as covalent apo(a)apoB complex (1) with a masked LBS II.
Double transgenic mice expressing both human apo(a) and human apoB-100
contain most of their apo(a) as components of Lp(a) particles (66) and should therefore represent a better model to
investigate the putative atherogenic properties of Lp(a). Our present
work is directed to use wild type and mutant Lp(a) transgenic animals
to evaluate a putative implication of the described LBS I and II for
the interaction of Lp(a) with components of the arterial wall.