© 1998 American Heart Association, Inc.
Original Contributions |
Sequences Within the Amino Terminus of ApoB100 Mediate Its Noncovalent Association With Apo(a)
From the Department of Biochemistry, Queen's University, Kingston, (B.R.G., M.L.K.), and the Lipoprotein and Atherosclerosis Research Group, Departments of Pathology and Laboratory Medicine and Biochemistry, University of Ottawa Heart Institute, Ottawa (R.S.M., Z.Y.), Ontario, Canada.
Correspondence to M.L. Koschinsky, PhD, Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
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Abstract—Although sequences within the C terminus of apolipoprotein B (apoB) have been implicated in the formation of covalent lipoprotein(a) [Lp(a)] particles, sequences in apoB that mediate initial noncovalent interaction with apo(a) remain to be characterized. To address this question, we have used an affinity chromatography method in which 2 recombinant forms of apo(a) [r-apo(a); either a 17-kringle form (17K) or a derivative containing apo(a) kringle IV types 5-8] have been immobilized onto Sepharose beads. Conditioned media from rat hepatoma (McA-RH7777) cell lines stably expressing various carboxyl-terminally truncated forms of human apoB (ranging from full-length apoB to apoB15) were applied to the r-apo(a) affinity columns; the columns were subsequently washed and eluted with
-aminocaproic acid (-ACA). Specific binding was quantified by
Western blot analysis of column fractions. Of the apoB
truncations examined, apoB94, apoB42, apoB37, and apoB29 exhibited
complete specific binding to 17K r-apo(a). Only 
50% binding was
observed for apoB18, whereas essentially no detectable binding was
observed with apoB15. In all cases, similar results were obtained when
the r-apo(a) kringle IV types 5-8–Sepharose column was used.
Additionally, substitution of proline for -ACA as the eluent
resulted in similar column profiles with either r-apo(a) affinity
column. We also demonstrated that apoB48 present in chylomicrons
bound completely to the 17K column in an -ACA–dependent manner.
Taken together, these results represent the first demonstration
that N-terminal sequences in apoB between amino acid
residues 680 (apoB15) and 781 (apoB18) are essential for noncovalent
association with apo(a) and that these sequences interact with
domain(s) present within apo(a) kringle IV types 5-8.
Key Words: lipoprotein(a) • apolipoprotein(a) • apolipoprotein B-100 • LDL • Lp(a) particle assembly
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Introduction |
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Lipoprotein(a) [Lp(a)] is a unique lipoprotein that closely resembles LDL both in lipid composition and the presence of the protein moiety apolipoprotein B100 (apoB). The distinguishing feature of Lp(a) is the presence of the highly glycosylated, polymorphic protein apolipoprotein(a) [apo(a)], which is covalently attached to apoB by a single disulfide bond.1 Lp(a) has been the focus of considerable research attention owing to a number of epidemiological studies that have identified elevated levels of Lp(a) as a risk factor for the development of atherosclerosis.2 3 However, the mechanism(s) by which Lp(a) contributes to the atherosclerotic process remains unclear.2 3
The process of Lp(a) assembly has been the subject of intensive investigation, with specific emphasis on the sequence requirements in both apo(a) and apoB that are required for Lp(a) formation. Several lines of evidence indicate that Lp(a) formation is predominantly an extracellular event: intracellular Lp(a) was undetectable in human liver homogenates,4 in the lysates of human hepatoma (HepG2) cells transfected with a 17-kringle (17K) form of recombinant apo(a) [r-apo(a)],5 or in cellular lysates of primary cultures of baboon hepatocytes expressing high levels of apo(a).6 Furthermore, there are recent data to suggest that Lp(a) assembly may occur on the hepatocyte cell surface.7 Although the aforementioned reports strongly suggest that Lp(a) formation occurs extracellularly, intracellular Lp(a) has been observed in HepG2 cells stably transfected with a 6-kringle form of r-apo(a).8
The generation of a number of recombinant expression systems for apo(a) has allowed for significant advances in our understanding of the process of Lp(a) formation. Studies examining Lp(a) assembly in vitro have led to the identification of inhibitors of the assembly process, including lysine, lysine analogues, and proline,9 10 as well as a thorough examination of the contribution of specific kringle domains of apo(a) to the process of Lp(a) formation. It is now well accepted that Lp(a) formation is a 2-step process in which the initial noncovalent association of apo(a) with the apoB moiety of LDL is mediated by the weak lysine-binding sites present in kringle IV types 5–8 (KIV5–8) of apo(a)10 11 12 and that this interaction likely precedes specific disulfide bond formation. Within this core sequence containing apo(a) KIV5–8, variable contributions of apo(a) kringle IV types 6, 7, and 8 in Lp(a) formation have been demonstrated.10 12 13 14
A relative paucity of information exists concerning the sequences in apoB that are necessary for Lp(a) formation. Examination of Lp(a) assembly in vitro by using carboxyl-terminally truncated forms of apoB expressed in the rat hepatoma cell line McA-RH7777 demonstrated that removal of the carboxyl-terminal 6% of apoB eliminated covalent Lp(a) formation.15 Moreover, plasma from mice expressing human apoB90 from a transgene did not support Lp(a) assembly in vitro, and no Lp(a) was detectable in mice transgenic for both apoB90 and apo(a).16 Taken together, these results suggested that sequences essential for Lp(a) formation, including the carboxyl-terminal cysteine of apoB (Cys4326), may reside within the carboxyl-terminal 6% of apoB. Site-directed mutagenesis of Cys4326 (in the context of full-length apoB) and subsequent expression of this mutant apoB species in mice have demonstrated that this cysteine residue is essential for both in vitro and in vivo Lp(a) formation.17 18 More recently, further studies using transgenic mice expressing the C-terminally truncated apoB derivatives apoB95 and apoB97 have shown that although apoB97 supports efficient Lp(a) formation, apoB95 assembles Lp(a) inefficiently both in vitro and in vivo.19 These findings suggest that amino acids 4331 to 4397 of apoB may directly contribute to the covalent association of apoB and apo(a) by allowing the proper positioning of the respective cysteine residues in these proteins that are involved in this process.
The observation that apoB95 supports Lp(a) assembly, albeit with greatly reduced efficiency, suggests that additional sequences contained within apoB95 are capable of mediating the noncovalent association of apoB with apo(a). To identify these sequences, we have examined the ability of a number of carboxyl-terminally truncated apoB variants contained in conditioned medium (CM) harvested from stably transfected McA-RH7777 cells to bind to r-apo(a) coupled to Sepharose 4B. Our results indicate that sequences between amino acids 680 (apoB15) and 781 (apoB18) are involved in noncovalent association with apo(a) and that these sequences bind specifically to 1 or more kringles present within apo(a) KIV5–8.
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Cloning and Expression of Recombinant Proteins
The details of the construction of the r-apo(a) derivatives 17K and KIV5–8 and the generation of human embryonic kidney (293) cell lines stably expressing these proteins have been previously described.20 21 The generation of expression plasmids encoding apoB100 and carboxyl-terminal truncations of apoB100, including apoB94,22 apoB42, apoB37, and apoB2923 as well as apoB1824 and apoB15,25 has been previously described as indicated. All apoB constructs were used to stably transfect the rat hepatoma cell line McA-RH7777 (American Type Culture Collection, Rockville, Md; No. CRL 1601) as previously reported. The organization of derivatives of apo(a) and apoB used in this study is shown schematically in Figure 1
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Human embryonic kidney (293) cells stably expressing either the 17K r-apo(a) or the r-apo(a) derivative KIV5–8 were routinely cultured in minimal essential medium (MEM, GIBCO/BRL) supplemented with 5% FBS (ICN). For the purpose of protein purification, confluent cell monolayers were maintained in serum-free medium (OptiMEM, GIBCO/BRL). CM was harvested and replaced every 48 hours and was stored at -20°C before use in protein purification (see below).
McA-RH7777 cells stably expressing apoB100 or C-terminal truncations of apoB were cultured in 100-mm dishes in the presence of MEM supplemented with 15% FBS. On reaching confluence, the medium was aspirated and replaced with 5 mL of serum-free MEM, and the cells were incubated at 37°C for 24 hours. At this time, the medium was harvested, clarified by centrifugation for 10 minutes at 1815g, and stored at -20°C.
Purification of r-Apo(a) Derivatives
r-Apo(a) derivatives were purified by affinity
chromatography by using lysine–Sepharose CL-4B
(Pharmacia) and a previously described
procedure.21 CM harvested from stably expressing
cell lines was loaded onto 50-mL lysine–Sepharose CL-4B columns
equilibrated with PBS, pH 7.4. Columns were washed with PBS containing
0.5 mol/L NaCl, pH 7.4, and eluted with 0.2 mol/L -aminocaproic acid
(-ACA) in the same buffer. Protein-containing fractions were pooled
and dialyzed extensively against HEPES-buffered saline (20 mmol/L
HEPES, pH 7.4, containing 0.15 mol/L NaCl). The dialyzed protein was
concentrated against PEG-20 000 (Fluka). Protein concentrations were
determined by measurement of absorbance at 280 nm using
corresponding molar extinction coefficients determined by the method of
tyrosine difference spectroscopy.26 Aliquots of
the purified proteins were stored at -70°C before use.
Isolation of LDL
Whole blood obtained from a normolipidemic volunteer was
collected into EDTA (final concentration, 1 mmol/L). Plasma was
isolated by low-speed centrifugation of whole blood
(710g for 15 minutes) and supplemented with 1 mmol/L
PMSF. Plasma was then adjusted to a density of 1.02 g/mL with NaBr and
centrifuged for 20 hours at 30 000 rpm (10°C) in a Beckman
60 Ti rotor. The infranatant was collected and the density adjusted to
1.063 g/mL with NaBr and recentrifuged as described above. The
floating LDL was harvested and recentrifuged at a density of
1.063 g/mL for a further 20 hours at 30 000 rpm. The floating LDL was
removed and passed over a Bio-Rad 10DG desalting column equilibrated
with PBS. The protein concentration was determined by measuring the
absorbance at 280 nm of the pooled LDL-containing fractions. The
isolated LDL was found to be devoid of contaminating proteins by
SDS–polyacrylamide gel electrophoresis (PAGE)
analysis.
Isolation of Chylomicrons
Whole blood was collected into EDTA (final concentration, 1
mmol/L) from a normolipidemic volunteer 90 minutes after ingestion of a
fatty meal. Plasma was isolated by low-speed
centrifugation of whole blood (710g for 15
minutes) and supplemented with 1 mmol/L PMSF. Plasma was then
centrifuged for 30 minutes at 20 000 rpm in a 70.1 Ti rotor
(Beckman) at 4°C. The floating chylomicrons were removed and
centrifuged again under the same conditions as described above.
The supernatant from this latter spin was isolated and concentrated by
centrifugation for 2 hours at 100 000 rpm in a Beckman
TL-100 rotor at 4°C. The floating chylomicrons were removed and the
protein content determined by using a modified Bradford assay (Bio-Rad)
with BSA (Sigma) as the standard.
Immobilization of r-Apo(a) Derivatives Onto Sepharose 4B
CNBr-activated Sepharose 4B (1 g, Pharmacia) was
resuspended in 1 mmol/L HCl and washed extensively with 200 mL of
1 mmol/L HCl in a sintered glass funnel. The washed resin was
resuspended in 5 mL of coupling buffer (0.1 mol/L
NaHCO3, pH 8.3, containing 0.5 mol/L NaCl).
Purified r-apo(a) (2 mg) corresponding to either the 17K or
KIV5–8 derivative was added to 1 mL of washed
gel and incubated overnight at 4°C. The slurry was washed 3 times
with 5 mL of coupling buffer to remove uncoupled r-apo(a), and any
remaining active groups were blocked by incubation of the coupled gel
resin overnight at 4°C with 10 mL of 0.1 mol/L Tris-HCl, pH 8. The
following day the gel was washed extensively with PBS containing 0.02%
NaN3 and stored at 4°C. Under the conditions
described above, >95% of each of the r-apo(a) derivatives was
immobilized onto the Sepharose 4B resin.
Recombinant Apo(a)–Sepharose 4B Affinity Chromatography
CM (0.25 mL) harvested from the apoB100 or carboxyl-terminally
truncated apoB cell lines was applied to 1.0 mL r-apo(a)–Sepharose
columns corresponding to either the 17K form of r-apo(a) or the
r-apo(a) derivative KIV5–8. The CM was allowed
to adsorb to the r-apo(a)–Sepharose columns for 30 minutes at room
temperature. At this time, the flow-through was collected and the
columns were washed with 5 to 6 mL of PBS containing 0.5 mol/L NaCl,
and 1-mL fractions were collected. Specifically bound protein was then
eluted with either 0.2 mol/L -ACA or 0.2 mol/L proline dissolved in
PBS containing 0.5 mol/L NaCl, and 1-mL fractions were collected. For
immunoprecipitations, all column fractions were incubated overnight at
4°C with 10 µg of a sheep polyclonal antibody raised against human
LDL (Calbiochem). Protein A–Sepharose (Pharmacia) was then added to
the samples and the mixtures were incubated for a further 30 minutes at
4°C. The Sepharose was then pelleted by brief
centrifugation at 16 000g and washed twice
with 500 µL of RIPA buffer (50 mmol/L Tris-HCl, pH 7.4; 150
mmol/L NaCl; 1% Triton X-100; 20 mmol/L EDTA; 0.5% sodium
deoxycholate; and 0.1% SDS)5 containing 0.5
mol/L NaCl and once with 500 µL of PBS. The pellets were then
resuspended in 2x Laemmli sample buffer27
containing 10 mmol/L DTT. Samples were boiled for 5 minutes, and
solubilized proteins were subjected to SDS-PAGE and Western blot
analysis as described below.
Isolated chylomicrons were diluted to a concentration of 25 µg/mL
with PBS, and 250 µL was adsorbed to the r-apo(a)–Sepharose 4B
columns for 30 minutes at room temperature. The columns were washed
with 6 volumes of PBS containing 0.5 mol/L NaCl and eluted with 0.2
mol/L -ACA in this buffer. Column fractions were immunoprecipitated
with 10 µg of a sheep polyclonal antibody raised against human LDL as
described above. The resulting immune complexes were resolved by
SDS-PAGE on a 6% polyacrylamide gel under reducing conditions
and subjected to Western blot analysis as described below.
In control experiments, purified LDL or BSA (100 µg each) was
chromatographed over Sepharose 4B columns to which either no
protein (mock) or the r-apo(a) derivatives 17K or
KIV5–8 had been immobilized as
described above. In all cases, the columns were washed with PBS
containing 0.5 mol/L NaCl (6x 1-mL washes), and specifically bound
protein was eluted from the columns with this buffer supplemented with
0.2 mol/L -ACA (6x 1 mL). The protein concentration in each
fraction was determined spectrophotometrically by measurement of the
absorbance at 280 nm. In chromatography experiments
using the apoB derivatives, each derivative was assessed for its
ability to bind to mock Sepharose columns; no binding was observed in
all cases.
Western Blot Analysis
Immunoprecipitates of r-apo(a) affinity column fractions were
electrophoresed on SDS-PAGE gels (5%, 6%, or 7.5%
polyacrylamide gels, depending on the size of the apoB species
being examined) under reducing conditions. After electrophoresis the
gels were transferred onto Immobilon P (ICN) membranes at 100 V for 1
hour in transfer buffer containing 25 mmol/L Tris-HCl, 192
mmol/L glycine, and 20% methanol. Membranes were blocked overnight at
room temperature with a 6% solution of skim milk powder dissolved in
NET buffer (50 mmol/L Tris-HCl, pH 7.4, containing 0.15 mol/L
NaCl, 50 mmol/L EDTA, and 0.05% Triton X-100). Blocked membranes
were probed with the apoB monoclonal antibody 1D1 (kind gift of Dr Ross
Milne, Ottawa Heart Institute) (200 ng/mL in NET) for 1 hour and then
washed extensively with several changes of NET over the course of an
hour. Membranes were then incubated for 1 hour with a horseradish
peroxidase–conjugated anti-murine IgG raised in sheep (Amersham,
1:5000 dilution in NET). The membrane was then washed with NET, and
immunoreactive proteins were detected with an enhanced
chemiluminescence (ECL) kit (Amersham) according to the manufacturer's
recommendations. Densitometric analysis of the resulting
immunoblot was performed by using a Hewlett-Packard Scanjet
3c flatbed scanner and analyzed by using Corel Photopaint
(version 7.0, Corel Corp) and Sigmagel (version 1.0, Jandel Scientific)
software. The percentage of apoB specifically eluted by -ACA or
proline was determined by dividing the sum of the density of -ACA or
proline fractions by the total amount of apoB (either full-length or
C-terminally truncated species) observed. For each apoB
truncation, 2 independent column chromatography
experiments were performed with each of the 17K- and
KIV5–8-Sepharose columns.
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Results and Discussion |
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To identify regions in apoB that mediate the first step of Lp(a) assembly, we have compared the ability of a number of C-terminally truncated apoB derivatives (Figure 1
) to bind
noncovalently to affinity columns containing either
immobilized 17K or KIV5–8 r-apo(a)
species (Figure 1). We utilized the KIV5–8
derivative in these experiments on the basis of recent studies in which
we have shown that core sequences within apo(a)
KIV5–8 directly mediate the -ACA–dependent
noncovalent interaction of apo(a) with apoB.28 To
validate our experimental system, we initially demonstrated that LDL
isolated from human plasma bound completely to either 17K- or
KIV5–8-Sepharose columns, whereas no detectable
binding was observed with BSA (Figure 2).
In each case, by the direct measurement of protein in column fractions,
we determined that the recovery of LDL and BSA from the columns was
>95% of the total protein applied (Figure 2). This suggests that we
can achieve quantitative recovery of the material applied to the
columns under these conditions, and this is supported by the
observation that treatment of the columns with the denaturant 0.1 mol/L
glycine (pH 2.3) did not result in the removal of any additional
protein (see below).
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In the current study, we observed that apoB100 present in the
CM harvested from stably transfected McA-RH7777 cells was able to bind
completely to a 17K r-apo(a)–Sepharose affinity column in the presence
of 0.5 mol/L NaCl. Furthermore, we found that the bound apoB100 could
be specifically eluted from the 17K affinity column by the addition of
the lysine analogue -ACA (Figure 3);
no additional protein could be eluted from the column by the addition
of 0.1 mol/L glycine (pH 2.3) (data not shown). Interestingly, a
protein corresponding in size to apoB48, which arises from a poorly
characterized posttranscriptional mechanism present in McA-RH7777
cells,22 also bound completely to 17K Sepharose
in the presence of 0.5 mol/L NaCl and could be completely eluted by the
addition of 0.2 mol/L -ACA (Figure 3). Identical elution patterns
were obtained for both apoB100 and the apoB48-like protein when CM
harvested from the stably expressing apoB100 cell line was
chromatographed over a KIV5–8
r-apo(a)–Sepharose affinity column (the Table
and data not shown).
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The ability of the apoB48-like species to bind to the
KIV5–8 affinity column demonstrates that
sequences in the N-terminal 48% of apoB are sufficient for
noncovalent association with core sequences present in apo(a)
KIV5–8, which is characterized by sensitivity of
this interaction to the addition of -ACA.10 11
To exclude the possibility that the observed noncovalent association of
the apoB48-like species was the result of either sequence differences
relative to plasma-derived apoB4822 or
differences in the composition of the lipid core, we assessed the
binding of chylomicrons isolated from human plasma to either the
KIV5–8 or 17K r-apo(a) affinity column. As shown
in Figure 4, chylomicron-derived apoB48
bound completely to the 17K Sepharose column, and the specifically
bound material was eluted from the column by the addition of -ACA.
Additionally, apoB100 corresponding to contaminating VLDL present
in the chylomicron preparation also bound completely to this column
(see Figure 4); similar results were obtained with VLDL purified from
human plasma by density gradient ultracentrifugation
(data not shown). Chylomicron-derived apoB48 also bound completely to
the KIV5–8 column in an -ACA–dependent
manner (the Table and data not shown). Taken together, these results
suggest that sequences present within the amino-terminal half of
apoB are sufficient to mediate complete noncovalent association with
apo(a) and that this binding interaction is mediated by sequences
within apo(a) KIV5–8. Additionally, our data
suggest that the noncovalent association of apoB and apo(a) is not
affected by the composition of the apoB48-containing particle with
respect to triglyceride and cholesteryl ester content.
Whereas chylomicrons are triglyceride-rich, cholesteryl
ester–poor particles of density <1.006 g/mL, the apoB48-containing
particles secreted by McA-RH7777 cells are smaller in diameter,
triglyceride poor, and present at densities between
1.10 and 1.19 g/mL.22 Clearly, the
presentation of sequences in apoB that interact
noncovalently with apo(a) is not affected by the size or lipid
composition of the apoB-containing particles.
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To exclude the possibility that the observed binding of apoB48 to
apo(a) was due solely to conformational constraints specific to apoB48,
we assessed the binding of the apoB94 species that lacks the
C-terminal 6% of apoB. As shown in Figure 5, apoB94 present in the CM harvested
from stably transfected McA-RH7777 cells bound specifically to 17K
Sepharose. ApoB94 present in the CM also bound completely to the
KIV5–8 affinity column and could be specifically
eluted with -ACA (the Table and data not shown). These results
demonstrate that despite its inability to form covalent Lp(a)
particles,15 apoB94 is able to associate
noncovalently with both r-apo(a) derivatives, thereby providing further
evidence for the role of amino-terminal sequences in apoB in mediating
its interaction with apo(a).
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To more precisely define the sequences within the amino-terminal half
of apoB that mediate noncovalent association with apo(a), we assessed
the noncovalent binding of other carboxyl-terminally truncated apoB
species (represented schematically in Figure 1) present
in the CM of stably transfected cells to either 17K or
KIV5–8 affinity columns. Both apoB42 and apoB37
bound specifically and completely to the 17K Sepharose and
KIV5–8 Sepharose (the Table and data not shown).
Although the apoB29 species bound completely to the 17K affinity
column, a fraction (15%) did not bind the
KIV5–8 Sepharose column (Figure 6 and the Table). Interestingly, with the
apoB18 species, we observed an apparent 50% decrease in specific
binding to either the 17K or KIV5–8 column,
whereas no specific binding to the r-apo(a) columns was observed with
CM containing the apoB15 species (Figure 6 and the Table). It appears
that the reduced binding of apoB18 to the r-apo(a) columns relative to
apoB29 reflects a functionally distinct pool of apoB18 that does not
bind to the column, as opposed to a reduced binding affinity of apoB18
for the immobilized apo(a). This was determined by
reapplication of the unbound apoB18 fraction to the 17K r-apo(a)
column; we observed that the apoB18 that did not bind to the column in
the first passage also showed no binding when it was reapplied to the
same r-apo(a) column (data not shown). No additional apoB18 could be
removed from the r-apo(a) column on the addition of denaturant (0.1
mol/L glycine, pH 2.3) (data not shown). The nature of the apoB18
species that shows no binding to the apo(a) column is unclear at
present. It is possible that the nonbinding pool of apoB18 may
differ in conformation or in glycosylation relative to the binding
fraction. No difference in electrophoretic mobility on a variety of gel
systems was observed (data not shown).
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Our observation that apoB15 did not bind at all to either 17K or KIV5–8 r-apo(a) columns indicates a drastically reduced affinity of this variant for apo(a). Taken together, our data suggest that sequences contained within a region corresponding approximately to amino acids 680 to 781 of apoB18 mediate its noncovalent association with apo(a) and that these sequences interact directly with sequences present within KIV5–8 of apo(a). It should be noted that this result is not an artifact of the column system used in the current study, since we also observed that apoB18, but not apoB15, could be coimmunoprecipitated with 17K r-apo(a) by a monoclonal anti-apo(a) antibody (data not shown). This suggests that the interactions observed on using r-apo(a)–Sepharose affinity chromatography also occur when apo(a) and apoB are free in solution.
To further characterize the interaction of apo(a) with the
C-terminally truncated apoB variants, we examined whether
proline, a known inhibitor of the in vitro formation of
Lp(a), could elute specifically bound truncated apoB species from
either of the 2 r-apo(a) affinity columns. As shown in Figure 7, both apoB42 and apoB18 could be
specifically eluted from KIV5–8-Sepharose by
proline; identical results were obtained with the 17K-Sepharose column.
These data, taken together with those obtained with the use of -ACA
for column elution, indicate that both proline and -ACA disrupt the
noncovalent association of the apoB variants with apo(a) by binding to
1 or more of the kringle pockets within KIV5–8.
Furthermore, since we have found that the specific binding of 17K and
KIV5–8 to LDL immobilized onto
microtiter wells can be abolished by the addition of either -ACA or
proline,28 the present study strongly
suggests that amino acids 680 to 781 of apoB may in fact contribute to
the high-affinity noncovalent binding of apo(a) and apoB that precedes
specific disulfide bond formation.
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Previous studies by our group15 and others16 have demonstrated that the C-terminal 6% to 10% of apoB is required for the formation of covalent Lp(a) particles. In the context of our current study, however, we find that apoB94 is capable of noncovalent association with r-apo(a). This suggests that lack of covalent Lp(a) formation when using this species arises owing to a defect in the second step of Lp(a) assembly involving disulfide bond formation. This is likely due to the absence of Cys4326 in apoB, which has been shown to be the cysteine residue in apoB that is responsible for disulfide bond formation with apo(a).17 18 Additionally, apoB94 lacks additional C-terminal sequences located between apoB95 and apoB97 that have been reported to be essential for efficient Lp(a) formation both in vitro and in vivo in transgenic mouse models.19 These latter sequences may be potential sites for the interaction of apo(a) KIV9 and apoB, which is necessary for disulfide bond formation, or may affect the presentation of Cys4326 for disulfide bond formation.
In conclusion, the current study clearly demonstrates a novel role for sequences in the amino terminus of apoB in mediating its noncovalent binding with apo(a). Since the noncovalent interaction of apo(a) and apoB is almost certainly a prerequisite for the formation of covalent Lp(a) particles, the sequences that we have identified in this study can be expected to play an important role in Lp(a) assembly in vivo. Clearly, future studies aimed at identifying specific amino acid(s) in apoB that are essential in mediating this noncovalent interaction will be of interest, particularly in the context of developing therapeutic strategies aimed at lowering plasma Lp(a) levels by inhibiting Lp(a) assembly.
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Acknowledgments |
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This work was supported by the Medical Research Council of Canada. B.R.G. is the recipient of a Research Traineeship from the Heart and Stroke Foundation of Canada. M.L.K and Z.Y. are Research Scholars of the Heart and Stroke Foundation of Canada.
Received January 9, 1998; accepted April 30, 1998.
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