© 1996 American Heart Association, Inc.
Articles |
Lys and Fibrinogen Binding of Wild-Type (Trp72) and Mutant (Arg72) Human Apo(a) Kringle IV-10 Expressed in E coli and CHO Cells
From the Departments of Medicine (O.K., A.M.S.) and of Biochemistry and Molecular Biology (A.M.S.) and the Committees on Genetics (A.M.S.) and Nutrition (A.M.S.), University of Chicago, Chicago, Ill.
Correspondence to Dr Olga Klezovitch, Department of Medicine, University of Chicago, 5841 S Maryland, MC5041, Chicago, IL 60637. E-mail oklezovi@medicine.bsd.uchicago.edu.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Abstract In a previous study, we identified a lysine (Lys)-binding–defective form of human lipoprotein(a) and attributed this defect to the presence of a Trp72
Arg mutation in
apolipoprotein(a) [apo(a)] kringle IV-10. To document this
relationship, we expressed both wild-type (wt) and mutant (mut)
forms of kringle IV-10 in Escherichia coli (nonglycosylated
form) and Chinese hamster ovary (CHO) cells (glycosylated form). The
Arg72 mut was prepared by introducing the TA mutation in apo(a)
kringle IV-10 amplified from human liver mRNA by the
reverse-transcriptase polymerase chain reaction technique. All
expressed kringles were tested for their ability to bind Lys and
plasmin-modified fibrinogen (PM-fibrinogen). wt kringle IV-10
expressed in both E coli and CHO cells bound to
Lys-Sepharose with comparable affinity. In contrast, the Arg72 mut
expressed in both systems exhibited no Lys-binding capacity.
Moreover, the wt kringle IV-10 expressed in both systems bound to
PM-fibrinogen and exhibited two binding components, one Lys mediated
(inhibitable by 
-amino-n-caproic acid) and one Lys
insensitive, occurring in about the same proportions. Only the latter
type of binding was present in the Arg72 mut expressed in E
coli. We conclude that kringle IV-10 of human apo(a) has
Lys-and PM-fibrinogen–binding capacities that are independent
of glycosylation and require the presence of Trp72, one of the seven
amino acids that constitute the Lys-binding site of kringle IV-10.
Our results also show that the binding of kringle IV-10 to
PM-fibrinogen is more complex than that to Lys, in that the former
requires an additional binding site or sites outside the
Lys-binding site.
Key Words: lysine and fibrinogen binding • Lp(a) • apo(a) kringle IV-10
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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One of the properties of human Lp(a) is its ability to bind Lys,1 a property that is not exhibited by the Lp(a) counterpart in rhesus monkeys.2 Although the molecular basis of this functional difference has not been clearly established, sequence analysis and modeling data3 have suggested a relationship between the Lys-binding defect and the presence of a Trp72
Arg mutation
in rhesus apo(a) kringle IV-10.2 Furthermore, we have
found the same mutation in humans with Lys binding–defective Lp(a)
by examining the DNA region that codes for the LBS of kringle
IV-10.4 The LBS of apo(a) kringle IV-10 comprises three
aromatic (Trp72, Trp62, and Phe64), two anionic (Asp55 and Asp57), and
two cationic (Arg35 and Arg71) amino acid residues (see Reference 3 for
a review). This LBS is identical to that of plasminogen
kringle 4, except that in the latter, Lys has replaced Arg in position
35. The importance of kringle IV-10 in Lys binding has also emerged
from recent studies of human apo(a) kringle IV-10 expressed in
Escherichia coli5 and human kidney embryonic
cells6 and of human apo(a) recombinants expressed in HepG2
cells.7 On the other hand, Rouy et al8
observed that the binding of recombinant apo(a) to fibrin was not
inhibited by the isolated kringle 4 of plasminogen and
concluded that human apo(a) kringle IV-10 had no affinity for the Lys
residues of fibrin. This same conclusion was reached by Sangrar et
al6 from studies of recombinant kringle IV-10 secreted
into the medium of transfected human kidney embryonic cells. Prompted
by these divergent observations and our general interest in
establishing a relationship between kringle structural mutability and
function, we performed studies of both wt Trp72 and mut Trp72Arg
forms of this kringle that were expressed in E coli and CHO
cells. The immediate goal of the study was to directly assess the role
of Trp72 in Lys and fibrin binding of the individual kringle IV-10
sequences and the influence of the carbohydrate moiety on Lys/fibrin
binding. The results of these studies are the subject of this
report. |
Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Expression of wt and mut Apo(a) Kringle IV-10 in E coli
PCR Cloning and Mutagenesis
All enzymes were obtained from New England Biolabs unless specified otherwise. The human liver was obtained from the Liver Transplant Program of the University of Chicago. Liver total RNA was isolated by use of the GlassMAX RNA microisolation kit (GIBCO BRL) and converted to cDNA by use of the SUPERSCRIPT preamplification kit (GIBCO BRL). The DNA region that codes for kringle IV-10 and spans nucleotides 12 383 through 12 725 of the apo(a) sequence9 was amplified by using primers A and B (Table
). PCR conditions in a reaction volume of 100 µL
were as follows: 200 ng human liver cDNA, 20 pmol of each primer, 200
µmol/L dNTP's, 1 U Vent Polymerase, denaturation at 94°C for 45
seconds, annealing at 57°C for 45 seconds, and extension at 72°C
for 1 minute for a total of 35 cycles. For all PCR reactions described
in "Methods," the reaction volume and concentrations of primers,
dNTP's, and Vent Polymerase were the same as stated above. The
resulting PCR product (342 bp) was cloned into the EcoRV
site of the pBlueScript KS- plasmid (Stratagene) and
used to transform DH5
cells (GIBCO BRL) according to the standard
techniques of Sambrook et al.10 Sequence analysis
of the isolated plasmid DNA revealed that all positive clones contained
Met in position 66 of kringle IV-10 (as originally reported by McLean
et al9 ) and not Thr, which is known to be a frequent
mutation in humans.11 12 13 The point
mutation TA that
results in substitution of Trp by Arg at position 72 of kringle IV-10
was introduced into the wt kringle sequence by using recombinant PCR
techniques.14 As a first step, we generated two fragments
that overlapped in sequence and contained the TA mutation as part of
amplification primers D and E (Table). The first PCR fragment
was
amplified with primers C and D; for the second fragment we used primers
E and F (Table). Primer C contained an EcoRI site,
whereas
primer F had the stop codon directly followed by an Xho I
site. The following PCR conditions were the same for both fragments:
500 ng pBlueScript/KIV-10, denaturation at 94°C for 30 seconds,
annealing at 57°C for 30 seconds, and extension at 72°C for 45
seconds for a total of 20 cycles. The PCR products were purified by
using a QIAEX Gel Extraction kit (Qiagen), and the purified fragments
were joined by reamplification with primers C and F. The conditions for
the reaction were as follows: 200 ng of each fragment, denaturation at
94°C for 30 seconds, annealing at 57°C for 30 seconds, and
extension at 72°C for 45 seconds for a total of five cycles, followed
by denaturation at 94°C for 45 seconds, annealing at 63°C for 45
seconds, and extension at 72°C for 1 minute for a total of 10 cycles.
The DNA fragment that codes for wt (Trp72) kringle IV-10 was amplified
directly from pBlueScript/KIV-10 by using primers C and F under the
following PCR conditions: 500 ng of plasmid DNA, denaturation at 94°C
for 30 seconds, annealing at 57°C for 30 seconds, and extension at
72°C for 45 seconds for a total of five cycles, followed by
denaturation at 94°C for 45 seconds, annealing at 63°C for 45
seconds, and extension at 72°C for 1 minute for a total of 15 cycles.
Finally, the DNA fragments that code for wt and mut kringle IV-10 were
gel purified and cloned into EcoRI- and Xho
I–digested vector pGEX-KG driven by the tac promoter and
containing the coding sequence for GST.15 The expression
plasmids were designated pGEX-KG/Trp72 KIV-10 and pGEX-KG/Arg72 KIV-10.
DNA sequences of wt and mut kringles were determined by using the fmol
Sequencing Kit according to the manufacturer's instructions
(Promega).
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Expression, Purification, and Thrombin Cleavage
of Fusion Apo(a)
Kringle IV-10
wt and mut forms of apo(a) kringle IV-10 were expressed
in
E coli strain DH5 as fusion GST-proteins according to the
instructions provided by Pharmacia. The GST–kringle IV-10 fusion
proteins bound to GST-Sepharose (Pharmacia) were cleaved by thrombin
for 4 hours at 22°C. The kringles were eluted in the flow-through
fraction, whereas the GST fusion component remained bound to the
column.
Analysis of free sulfhydryl groups in recombinant kringle IV-10 was performed according to the method of Ampulski et al.16 Kringle IV-10 (20 µmol/L) was exposed to a six-molar excess of 4,4'-dithioldipyridine, and the reaction was monitored at 324 nm for 20 minutes at room temperature.
Expression of wt and mut Kringle IV-10 in CHO Cells
Plasmids and Amplification Primers
Plasmid
pBlueScript/KIV-10 was used as a template for generating
wt (Trp72) and mut (Arg72) kringle IV-10 expression constructs as
described in the previous section, except that primer C was replaced
with primer G containing a BsoFI site (Table
). The PCR
products were digested with BsoFI and Xho I
and then gel purified. The human apo(a) leader (95 bp), containing part
of the 5' untranslated region and the signal peptide, was obtained by
digestion of cloned apo(a) DNA (PUC119Da18.4 plasmid, generously
provided by Dr J.W. McLean) with EcoRI and BsoFI.
Apo(a) leader DNA was gel purified, ligated to DNA fragments that code
for either wt or mut kringle IV-10, and inserted into cytomegalovirus
promoter–driven mammalian expression vector pcDNA3 (Invitrogen)
restricted with EcoRI and Xho I. The expression
plasmids were designated pcDNA3/Trp72 KIV-10 and pcDNA3/Arg72 KIV-10.
DNA sequences of both wt and mut constructs were determined as
described above.
Tissue Culture and Transfection
Experiments
CHO cells, generously provided by Dr G. Dawson (University
of
Chicago, Chicago, Ill), were cultured at 37°C in the presence of 10%
CO2 with DMEM-F12 medium (GIBCO BRL) supplemented with 10%
fetal bovine serum (GIBCO BRL). The expression plasmids pcDNA3/Trp72
KIV-10 and pcDNA3/Arg72 KIV-10, purified by use of the Plasmid Midi Kit
(Qiagen), were combined with Lipofectin reagent (GIBCO BRL);
transfection was performed according to the GIBCO BRL protocol. Two
days after transfection, the cells were plated in DMEM-F12 medium
supplemented with 10% fetal bovine serum and 400 µg/mL geneticin
(G418, GIBCO BRL). After 10 days stable, individual clones were
identified. Several colonies were analyzed, and the clones that
were producing the largest amount of recombinant kringles were selected
for further examination.
Partial Purification of wt Kringle
IV-10 From the Culture
Media
Trp72 kringle IV-10 secreted by the stable transformants was
isolated from conditioned media by affinity
chromatography on Lys-Sepharose (for details, see the
"Lys-Sepharose Binding Assay" section). The cells were washed
twice with serum-free DMEM-F12 and incubated with the same medium
overnight at 37°C. The medium (200 mL) was batch adsorbed with 5 mL
Lys-Sepharose for 2 hours at 22°C. The resin was then packed in the
column and washed with 10 bed volumes of PBS followed by 10 bed volumes
of the same buffer containing 0.5 mol/L NaCl. Bound proteins were
eluted with PBS in the presence of 0.2 mol/L EACA (Sigma). The
kringle-containing fractions were combined and dialyzed against 50
mmol/L Tris HCl, pH 7.5/0.15 mol/L NaCl.
In Vitro Translation
In vitro translation was performed using
the TNT/T7 coupled
reticulocyte lysate system and canine pancreatic microsomes from
Promega. Aliquots (1 µg) of pcDNA3/KIV-10 (either wt or mut) and
L-[35S]Cys (DuPont-NEN) were added to the
reaction mixture, and translation was performed according to the
manufacturer's protocol. The proteins were separated by 15%
SDS-PAGE17 using a Mini-Gel electrophoresis system from
Novex. The gels were treated with En3hancer
Autoradiography Enhancer (DuPont-NEN), dried at
60°C under vacuum, and exposed to the film at -70°C.
Radiolabeling Studies
Steady-State Labeling Experiments
CHO cells (80% confluent) were preincubated for 1 hour at
37°C in serum-free Met/Cys–depleted DMEM (GIBCO BRL) and
then labeled for 5 hours in the same medium supplemented with
Expre35S35S label containing both
[35S]Met and [35S]Cys (125
µCi/mL,
DuPont-NEN). The supernatants were harvested after the labeling period
and either studied immediately or frozen at -20°C until
use.
Pulse-Chase Experiments
After preincubation
for 1 hour at 37°C in serum-free
Met/Cys–depleted DMEM, the CHO cells were labeled for 10 minutes
with Expre35S35S label (250 µCi/mL) and
chased from 0 to 1 hour at 37°C in serum-free DMEM. The
supernatants were harvested and analyzed by
immunoprecipitation. Cell monolayers were rinsed twice with cold PBS
and scraped into a cold lysis buffer (50 mmol/L Tris HCl, pH 7.5; 0.15
mol/L NaCl; 5 mmol/L EDTA; 0.5% Triton X-100; 0.5% sodium
deoxycholate; and 1 mmol/L PMSF). The lysates were clarified by
centrifugation in an Eppendorf centrifuge for
10 minutes at 4°C and further analyzed by
immunoprecipitation.
Preparation of Anti–Kringle IV-10 Antibody
A polyclonal
antibody to human apo(a) kringle IV-10 was
prepared at the Animal Facilities of the University of Chicago. Rabbits
were injected with 0.5 mg wt human apo(a) kringle IV-10 expressed in
E coli that had been mixed in complete Freund's adjuvant
(GIBCO BRL) and at monthly intervals were given two more injections of
0.2 mg kringle mixed with incomplete Freund's adjuvant (Sigma).
Antiserum was collected and frozen at -20°C.
Immunoprecipitation
Metabolically labeled supernatants or
cell
lysates were incubated for 1 hour at 22°C with anti–apo(a)
kringle IV-10 serum (1 µL serum per 106 cpm).
Thereafter, protein A–Sepharose (Pharmacia) was added and the samples
were incubated for an additional 30 minutes at 22°C. The Sepharose
was pelleted by brief centrifugation and washed twice
with cold NETTAM buffer (50 mmol/L Tris HCl, pH 7.5; 0.15 mol/L NaCl; 5
mmol/L EDTA; 1 mg/mL BSA; 1% Triton X-100) followed by two additional
washes with cold RIPA buffer (50 mmol/L Tris HCl, pH 7.5; 0.65 mol/L
NaCl; 10 mmol/L EDTA; 1% Triton X-100; 1% sodium deoxycholate; 0.1%
SDS) and a final wash with cold water. Laemmli sample
buffer17 was then added and the samples were boiled for 5
minutes in the presence of 5% ß-ME (Bio-Rad). The solubilized
proteins were separated by 15% SDS-PAGE. The gels were treated with
autoradiography enhancer, dried at 60°C under
vacuum, and exposed to film at -70°C.
Lys-Sepharose Binding Assay
E coli–Expressed Kringles
Cyanogen
bromide–activated Sepharose (Pharmacia)
was coupled to the -amino group of L-Lys (Sigma)
essentially according to the company's instructions. The amount of Lys
cross-linked to the beads ranged between 16 and 21 µmol/mL of
bead suspension. Kringle IV-10 (50 µg in 0.2 mL PBS) was batch
incubated with 0.2 mL Lys-Sepharose for 15 minutes at 22°C. The
Sepharose was then washed three times with five bed volumes of PBS. The
bound protein was eluted with two bed volumes of 0.2 mol/L EACA in PBS,
and the resulting fractions were analyzed by 15% SDS-PAGE. The
proteins were visualized by staining the gels with Coomassie Brilliant
Blue R-250.
CHO Cell–Expressed Kringles
Aliquots (1 mL) of labeled, cell-culture supernatants (see
"Steady-State Labeling Experiments") were incubated with 0.2
mL Lys-Sepharose, as described for the E coli–expressed
kringles. The fractions were immunoprecipitated with anti–kringle
IV-10 serum and analyzed by SDS-PAGE, followed by fluorography
as described in the previous section.
PM Fibrinogen Binding Assay
Conditons for our binding assays
were adapted from those of
Harpel et al18 and LoGrasso et al.5
Ninety-six–well plates were incubated with 1 µg fibrinogen
per well (Sigma) in TBS buffer (50 mmol/L Tris HCl, pH 7.5; 0.15 mol/L
NaCl; and 0.2% BSA) for 2 hours at 37°C. After the wells were
emptied, 2% BSA in TBS buffer was added to the plates for 1.5 hours at
22°C. The wells were washed three times with TBST and further treated
with 3 ng plasmin per well (Enzyme Research Laboratories) for 40
minutes at 37°C. Plasmin was inactivated by incubating
the wells with TBST containing the protease inhibitor
p-nitrophenyl p'-guanidinobenzoate (Sigma) at a
final concentration of 0.1 mmol/L for 20 minutes at 22°C. After two
additional washes with TBST, various concentrations of either purified
kringle or cell culture medium that had been diluted with TBS with or
without 0.2 mol/L EACA were added and incubated overnight at 22°C.
Thereafter, the wells were washed three times with TBST, and after
addition of rabbit anti–apo(a) kringle IV-10 serum (1:500),
incubated for an additional hour at 22°C. The wells were then washed
four times with TBST, and the secondary antibody (goat anti-rabbit
IgG alkaline phosphatase conjugate [1:2000, Sigma]) was added for 1
hour at 22°C. After another four washes with TBST, the wells were
incubated with 1 mg/mL p-nitrophenylphosphate (Sigma), and
color development was monitored at 405 nm on a microplate reader
(Biomek 1000, Beckman). We derived
Kd values from the Langmuir equation, assuming
single-site binding according to Fleury and
Anglés-Cano.19
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Expression of wt and mut Apo(a) Kringle IV-10 in E coli
Both GST-Trp72 kringle IV-10– and GST-Arg72 kringle IV-10–expressed fusion proteins were found in the soluble fraction; thus, they could be readily purified by one-step chromatography on GST-Sepharose as described in "Methods." wt and mut kringles were then separated from the GST-protein by thrombin cleavage of the fusion protein bound to the column matrix. The product, containing the 112 amino acids of apo(a) kringle IV-10 and the 12 amino acids of GST-protein (GSPGISGGGGGI), was eluted in the flow-through fraction. The yields of purified kringles ranged from 0.4 to 0.8 mg/L of medium. By SDS-PAGE we estimated that the purity of the isolated kringles was >90% (Fig 1A
). On the SDS gel, both wt and mut forms of kringle
IV-10 migrated as proteins with an approximate molecular mass of
14 500 D (the predicted molecular masses for Trp72 kringle IV-10 and
Arg72 kringle IV-10 were 15 861 and 15 831 D, respectively). There
were no detectable free sulfhydryl groups in the expressed kringles on
the basis of their failure to react with
4,4'-dithioldipyridine, a Cys-specific modifying
reagent.16
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In Vitro Translation Experiments
Direct expression of
pcDNA3/Trp72 KIV-10 and pcDNA3/Arg72
KIV-10 plasmids in the in vitro translation system resulted in the
synthesis of proteins with apparent molecular masses of 17 000 D
(predicted molecular masses for Trp72 kringle IV-10 and Arg72 kringle
IV-10 containing the signal peptide were 17 239 and 17 209 D,
respectively; Fig 2, lane 1). To determine the
efficiency of the processing events (ie, signal peptide cleavage and
core N glycosylation of the kringles), in vitro translation
experiments were performed in the presence of different amounts of
microsomal vesicles (from 0 to 1.8 µL per 25 µL of reaction mix).
Analysis of the translation products by SDS-PAGE revealed
two additional proteins with apparent molecular masses of 18 500 and
19 000 D (Fig 2, lanes 2 through 4). Synthesis of these two
proteins
was completely abolished by the addition of tunicamycin (data not
shown) indicating that they represented the
N-glycosylated wt and mut forms of kringle IV-10.
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Synthesis and Secretion of wt and mut Apo(a) Kringle IV-10 by
CHO Cells
By Northern blot analyses, the sizes of the
transcripts corresponding to Trp72 kringle IV-10 and Arg72 kringle
IV-10 were estimated to be 
0.9 kb, as predicted (data not shown).
Our polyclonal antibody raised against E coli–expressed
Trp72 kringle IV-10 exhibited a high affinity for both wt and mut
glycosylated kringles during the in vitro translation experiments.
Thus, we used this antibody for direct immunoprecipitation of kringles
secreted by CHO cells. The cells were metabolically labeled
and the conditioned medium immunoprecipitated as described in
"Methods." Analysis of the immunoprecipitates by SDS-PAGE
under reducing conditions (Fig 1B) gave an apparent molecular
mass of
22 000 D for the secreted kringles. The calculated molecular masses
for Trp72 kringle IV-10 and Arg72 kringle IV-10 without signal peptide
were 14 632 and 14 602 D, respectively; those of the kringles that
had been glycosylated during in vitro translation experiments were
18 500 and 19 000 D, respectively. These data suggest that the
kringle IV-10 secreted by CHO cells into the conditioned medium was
also O-glycosylated.
To determine the effect of the
Trp72Arg mutation on the synthesis
and secretion rate of apo(a) kringle IV-10, cells from a
representative pool of three independent clones were
pulse-labeled for 10 minutes with [35S]Met and
[35S]Cys and chased for 0 to 1 hour. The cell lysates and
media were then immunoprecipitated with anti–apo(a) kringle IV-10
and analyzed by SDS-PAGE and fluorography. No difference in
secretion rate between wt and mut kringles was observed (Fig
3). After a 30-minute chase, both kringles were detected
in the medium. The two intracellular proteins (18 500 and 19 000 D)
presumably represent the N-glycosylated precursors
of kringle IV-10, by comparison of their masses with those of the in
vitro translated kringles (Fig 2).
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Lys-Binding Properties of Expressed Kringles
The Lys-binding
capacities of the recombinant Trp72 and
Arg72 IV-10 kringles were examined by affinity
chromatography on Lys-Sepharose as described in
"Methods." Both E coli– and CHO-expressed wt
kringle
IV-10 bound avidly to Lys-Sepharose, so only small amounts (<5%) of
the kringles were found in the flow-through fraction (Fig 4A
and 4B, lane 1); the bound kringles were then eluted with
0.2 mol/L EACA (Fig 4A and 4B, lane 3). In
contrast to the wt kringle,
both the E coli– and CHO-expressed Arg72 mut had no
affinity for Lys-Sepharose and was detected exclusively in the
flow-through fraction (Fig 4A and 4B, lane 1).
These results
provide direct evidence for the crucial role of Trp72 in the binding of
human apo(a) kringle IV-10 to Lys, indicating that the presence of
carbohydrates has no effect on this binding.
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Binding of Recombinant Kringles to PM-Fibrinogen
wt kringle
IV-10 expressed in E coli readily bound
to immobilized PM-fibrinogen (Fig 5A). This
binding had two components: one Lys mediated, inhibited by 0.2 mol/L
EACA and one non–Lys-mediated, unaffected by the presence of the Lys
analogue. The Lys-mediated binding was found to be saturable, with
an apparent Kd of 23±8 µmol/L. The
non–Lys-mediated component represented 60% of the
total binding of Trp72 kringle IV-10 to fibrinogen. No
Kd could be determined for this binding
component. In contrast, the Arg 72 mut expressed in E coli
exhibited only the non–Lys-mediated binding component (Fig
5B).
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) or
presence (
) of 0.2 mol/L EACA as described in "Methods."
Lys-mediated binding (
) was calculated by subtracting the
binding in the presence of 0.2 mol/L EACA from total binding. Each
point is the mean of three measurements.
The fibrinogen-binding test for Trp72 and Arg72
IV-10 kringles
expressed in CHO cells was performed in the presence of conditioned
media. The range of kringle concentrations in the
fibrinogen-binding assay was estimated to be the same as that for
purified kringles expressed in E coli, as determined by an
ELISA using anti–apo(a) kringle IV-10 antibody. Neither the wt nor
mut form of kringle IV-10 expressed in CHO cells exhibited binding to
immobilized PM-fibrinogen. We postulated that other
proteins in the CHO cell medium could have affected binding. In fact,
addition of medium from mock-transfected CHO cells to the purified
Trp72 kringle IV-10 expressed in E coli completely abolished
the binding of this kringle to immobilized PM-fibrinogen
(data not shown). Of interest, presence of the medium did not influence
the binding of wt kringle IV-10 expressed in CHO cells to
Lys-Sepharose. On the basis of this latter observation, we purified
Trp72 kringle IV-10 from the conditioned medium by affinity
chromatography on Lys-Sepharose (see "Methods")
and used this preparation in the fibrinogen-binding studies.
However, we were unable to use this method of purification for mut
Arg72 kringle IV-10 because of its inability to bind to Lys. The purity
of the isolated wt kringle was estimated to be 50% by SDS-PAGE on
silver-stained gels (data not shown). Kringle concentration was
measured by an ELISA using the anti–apo(a) kringle IV-10 antibody
against known concentrations of wt kringle IV-10 expressed in E
coli. Under our assay conditions, wt kringle IV-10 that had been
partially purified from the conditioned medium bound to
immobilized PM-fibrinogen (Fig 6). As with
wt kringle IV-10 expressed in E coli, the fibrinogen binding
of Trp72 kringle IV-10 expressed in CHO cells exhibited two
components: one Lys mediated, inhibited by 0.2 mol/L EACA, and
one non–Lys-mediated, unaffected by the presence of the Lys
analogue. The Lys-mediated binding component was saturable, with an
apparent Kd of 3.9±1.2 µmol/L, on the same
order of magnitude as that for Trp72 kringle IV-10 expressed in E
coli (23±8.7 µmol/L). The non–Lys-mediated component
represented 40% of the total binding of this kringle to
fibrinogen. No Kd could be determined for this
binding.
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To investigate the nature of the second, non–Lys-mediated binding component in apo(a) kringle IV-10, we performed PM-fibrinogen binding experiments in the presence of Pro, which was shown by Trieu et al20 to inhibit Lp(a) assembly and the binding of apo(a) to apoB-containing lipoproteins. In our studies at concentrations as high as 0.2 mol/L, Pro had no effect on the binding of apo(a) kringle IV-10 to PM-fibrinogen (data not shown).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The current investigation directly supports our previous proposal that human kringle IV-10 has the capacity to bind to Lys and fibrinogen and that Trp72 plays an essential role in this binding.2 4 In terms of Lys binding, our data are in accord with the crystallographic studies of plasminogen kringle 4 by Mulichak et al21 and with the work on recombinant apo(a)/Lp(a) expressed in HepG2 cells by Ernst et al.7 We can also conclude that neither Lys nor fibrinogen binding appears to be influenced by the Met66
Thr mutation, because
binding was observed with the Met66 wt kringle IV-10 in the current
investigation and with the Thr66 form studied by LoGrasso et
al.5 Fless and Snyder22 recently observed that the binding of plasminogen to thrombin-treated fibrin was mediated almost entirely by Lys residues, contrary to Lp(a) binding, which exhibited two components: one LBS mediated and the other LBS independent. In those studies the contribution of the LBS-independent component to the total binding varied from 33% to 80%, depending on the Lp(a) polymorph used.22 Our work now shows that the individual wt kringle IV-10 also binds to PM-fibrinogen via two components, one LBS mediated and another LBS independent. A similar observation concerning apo(a) kringle IV-10 was recently reported by LoGrasso et al,5 although the proportion of the LBS-independent component (referred to by the authors as "nonspecific") was significantly less than in our studies (15% versus 40% to 60%). In addition, our current investigation has documented the occurrence of this LBS-independent binding component in mut (Arg72) kringle IV-10, indicating that kringle IV-10 can bind to PM-fibrinogen even without a functional LBS. We currently have no knowledge of the mechanism(s) responsible for this "nonspecific" binding. We must note, however, that although the bimodal binding of apo(a) kringle IV-10 to PM-fibrinogen is qualitatively similar to that reported for Lp(a) binding to thrombin-treated fibrin,22 there are important quantitative differences in Kd values: micromolar for kringle IV-10 versus nanomolar for Lp(a). This disparity suggests that either there is a cooperative effect among kringles in terms of apo(a) binding to fibrin(ogen) and/or an additional site or sites on apo(a) are implicated in this binding. Recently, Huby et al23 reported that a "mini-Lp(a)," which was prepared by limited proteolysis of Lp(a) and composed of kringles IV-5 through IV-10, kringle V, and the protease region, bound to either intact or PM-degraded fibrin with a Kd similar to that of intact Lp(a). This finding contrasted with results obtained with the N-terminal domain of apo(a), which comprised kringle IV-1, several kringle IV-2 repeats, kringle IV-3, and kringle IV-4 and did not bind to fibrin either in the presence or absence of EACA.23 Therefore, the location of the additional fibrin-binding site(s) can be restricted to the C-terminal region of apo(a). In addition, Ernst et al7 have recently identified two functionally distinct LBSs on human apo(a), in kringle IV-10 (LBS I) and kringle IV-5–IV-9 (LBS II). However, the authors did not examine whether LBS I and/or LBS II was involved in fibrin binding.
In the current study we also demonstrated that glycosylation had no effect on the binding of recombinant kringle IV-10 to Lys and PM-fibrinogen. On the other hand, Sangrar et al6 observed no fibrinogen binding with recombinant apo(a) kringle IV-10 expressed in human embryonic kidney cells and examined in the presence of conditioned medium. It is likely that in those studies, the expressed kringle was functionally competent but unable to exhibit its binding capacity owing to interfering components in the cell medium. This possibility is supported by our results, which show that the medium of mock-transfected CHO cells inhibits the fibrinogen binding of wt kringle IV-10 expressed in E coli. Moreover, wt kringle IV-10 that had been partially purified from CHO cell medium readily bound to PM-fibrinogen. From these observations it is apparent that caution should be used in interpreting negative binding data from the study of impure products.
In a study concerning the interaction of a purified recombinant 17-kringle apo(a) with a PM-fibrin surface, Rouy et al8 found that the binding was not inhibited by plasminogen kringle 4 and interpreted their observation to suggest that apo(a) kringle IV-10 lacks fibrin-binding capacity. On the other hand, LoGrasso et al5 observed that recombinant apo(a) kringle IV-10 expressed in E coli did inhibit the binding of Lp(a) to immobilized PM-fibrinogen. Based on those observations and our own, it is evident that data obtained with plasminogen may not be readily extrapolated to the apo(a) system.
Taken together, our results indicate that the individual apo(a) kringle IV-10 has Lys- and PM-fibrinogen–binding capacities that are independent of glycosylation and require Trp72, one of the key amino acids for the LBS of this kringle. Our results have also shown for the first time that mut Arg72 kringle IV-10 lacks a functional LBS and therefore binds to PM-fibrinogen through a non–Lys-mediated component outside the LBS domain. This finding may be clinically relevant because it indicates that Lp(a), by binding to fibrin(ogen), may exhibit a prothrombotic action even in the absence of a functional kringle IV-10 LBS.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by National Institutes of Health–National Heart, Lung, and Blood Institute program project grant 18577 (Bethesda, Md). We thank Celina Edelstein for fruitful discussions during the course of the studies and Dr Glyn Dawson for providing the CHO cells.
Received June 29, 1995; accepted November 10, 1995.
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References |
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