From the Oxford Glycobiology Institute, Department of
Biochemistry, University of Oxford, South Parks Road,
Oxford OX1 3QU, United Kingdom and ¶ INSERM, Unite 551, Hopital de la Pitie, 75651 Paris, France
Received for publication, March 9, 2001, and in revised form, April 3, 2001
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ABSTRACT |
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Apolipoprotein(a) (apo(a)) is a
multikringle domain glycoprotein that exists covalently linked to
apolipoprotein B100 of low density lipoprotein, to form the
lipoprotein(a) (Lp(a)) particle, or as proteolytic fragments.
Elevated plasma concentrations of apo(a) and its fragments may promote
atherosclerosis, but the underlying mechanisms are incompletely
understood. The factors influencing apo(a) proteolysis are also
uncertain. Here we have used exoglycosidase digestion and mass
spectrometry to sequence the Asn (N)-linked and Ser/Thr
(O)-linked oligosaccharides of human apo(a). We also
assessed the potential role of apo(a) O-glycans in
protecting thermolysin-sensitive regions of the polypeptide. Apo(a)
contained two major N-glycans that accounted for 17% of the total oligosaccharide structures. The N-glycans were
complex biantennary structures present in either a mono- or
disialylated state. The O-glycans were mostly (80%)
represented by the monosialylated core type 1 structure,
NeuNAc Lipoprotein(a) (Lp(a))1
consists of a low density lipoprotein (LDL) particle covalently linked
through a single disulfide bond to apolipoprotein(a) (apo(a)) (1).
Apo(a) contains multikringle domains that are homologous to plasminogen
kringles IV and V (2). This homology is thought to underlie the
atherogenicity of Lp(a) because apo(a) and its naturally occurring
proteolytic fragments compete with plasminogen for fibrin(ogen) binding
(3, 4). The predicted apo(a)-mediated decrease in fibrinolysis may
contribute to the accumulation of fibrin at sites of atherosclerotic
lesion development. Apo(a) also exhibits additional proatherogenic
properties including binding to fibronectin (5) and decorin (6), direct inhibition of tissue-type plasminogen activator 1 action (7), and
modulation of fibrinolysis after cell-surface binding (8, 9).
The concentration of plasma and urinary apo(a) proteolytic fragments is
increased concomitantly with elevated plasma Lp(a) levels (10, 11). In
some cases, the apo(a) fragments have a higher atherogenic potential
than the intact Lp(a). For example, when the apo(a) moiety of Lp(a) is
cleaved by protease to generate "mini-Lp(a)" (12), the resulting
particle binds fibrinogen (12) and fibronectin (13) more avidly than
undigested Lp(a). Because apo(a) fragments have potentially atherogenic
properties and because they are also known to accumulate in
atherosclerotic lesions (14), it is important to understand the factors
that regulate apo(a) proteolysis. Previous studies have focused on the
role of serine proteases and metalloproteinase as relevant enzymes (5,
12, 15, 16); however, the factors related to the proteolytic
susceptibility of the various apo(a) domains have not been addressed.
The presence of O-glycans is known in some cases to decrease
the sensitivity of certain polypeptides to proteolytic cleavage (17).
Based on monosaccharide analysis, apo(a) is predicted to contain a high
degree of O-glycosylation (18), and the high content of Ser
and Thr residues in the interkringle linker domains suggested that
these were the most likely sites for glycan attachment (2, 19).
Furthermore, studies of apo(a) glycopeptides revealed that the
O-glycans are clustered in the kringle IV linker domains (20). This raised the possibility that apo(a) O-glycans
could restrict proteolytic cleavage of the interkringle linkers. In the present work we have used exoglycosidase digestion and mass spectrometry to elucidate the structures of apo(a) glycans, and we
investigated the potential importance of the major glycan species in
modulating the sensitivity of apo(a) to thermolysin digestion.
Materials--
Acetonitrile (chromosolv) and hexane were from
Riedel-de Haen, Haen, Germany. Methanol, chloroform, and KBr were from
BDH, Poole, UK. Acetone was from Fisher. Phosphate-buffered saline was
prepared using Oxoid (Basingstoke, UK) tablets. All reagents for
hydrazinolysis were from Oxford GlycoSciences, Abingdon, UK The
Atherobacter ureafaciens sialidase (EC 3.2.1.18, ABS) and bovine testes galactosidase (EC 3.2.1.23, BTG) were from Glyko (Novato, CA), Clostridium perfringens sialidase (EC
3.2.1.18, CPS) was from Sigma (catalog number N2133). All other
reagents were of the highest quality available and were purchased from Sigma.
Study Subjects--
The plasma lipid and Lp(a) concentrations of
the two male and one female subjects under study are given in Table
I. Blood was drawn on EDTA (0.1%) after
an overnight fast. Total cholesterol and triglycerides were determined
by nephelometry. Plasma Lp(a) concentration was determined by
enzyme-linked immunosorbent assay, and apo(a) isotyping was performed
as described previously (21).
Isolation and Purification of Lp(a) and Apo(a)--
Lp(a) and
apo(a) were isolated by sequential ultracentrifugation and
lysine-Sepharose affinity chromatography as described previously (12).
After purification, Lp(a) (1 mg/ml) was incubated with dithiothreitol
(10 mM) for 3 h at 37 °C. After this treatment, apo(a) was separated from the remaining LDL-like particles on heparin-Sepharose according to Ref. 11 using 1 mg of Lp(a) per 2 ml of
resin. All subsequent analyses were performed on individual Lp(a) or
apo(a) samples.
Release of N-Linked Glycans by Automated
Hydrazinolysis--
Purified apo(a) was subjected to automated
hydrazinolysis (22) using a GlycoPrep 1000 instrument (Oxford
GlycoSciences Ltd.). For the N-glycan release,
hydrazinolysis was performed at 100 °C for 5 h in order to
achieve maximal recovery. Since these conditions resulted in a partial
degradation of O-glycans via a Release of O-Linked Glycans by Manual
Hydrazinolysis--
Purified apo(a) was lyophilized and then
cryogenically dried before hydrazinolysis (26). Samples were incubated
with hydrazine for 6 h at 60 °C to release the
O-linked glycans (27). Excess hydrazine was removed by
evaporation, and the glycans were re-N-acetylated with
acetic anhydride in 0.2 M sodium acetate, pH 8.0. Sodium salts were removed with a column containing 5-fold binding excess of
Dowex AG50 × 12(H+) 200-400 mesh (Bio-Rad) followed
by elution with 5 volumes of water. Peptides were removed by descending
paper chromatography on Whatman 1MM chromatography paper in
butanol:ethanol:water (8:2:1 v/v/v) for 48 h. Glycans were
recovered from the paper ( Simultaneous Exoglycosidase Sequencing of the Released Glycan
Pool--
The 2-AB labeled glycan pools were evaporated to dryness,
and standardized enzyme solutions were added to individual aliquots of
each sample (28). The indicated mixtures were incubated for 16-24 h at
37 °C in 100 mM citrate:phosphate buffer (pH 5)
containing 0.2 mM zinc acetate and 0.15 M NaCl.
ABS and BTG were used at a concentration of 1-2 units/ml.
HPLC Systems--
Normal phase (NP) and reversed phase (RP) HPLC
separations were performed on Waters (Watford, UK) 2690 Alliance
separations modules equipped with Waters temperature control modules
and Waters 474 fluorescence detectors. External degassers were also
used (Douglas Scientific, Southampton, Hampshire, UK). Systems were controlled via Waters Millenium 32 software.
HPLC Conditions--
Normal phase HPLC was performed using a
4.6 × 250 mm GlycoSep-N column (Glyko, Novata, CA). Solvent A was
50 mM formic acid adjusted to pH 4.4 with ammonia solution.
Solvent B was acetonitrile. Column temperature was set to 30 °C.
Gradient conditions were as follows: t = 0 min,
flow = 0.4 ml/min, 20% solvent A; t = 152 min, flow = 0.4 ml/min, 58% solvent A; t = 155 min, flow = 0.4 ml/min, 100% solvent A; t = 157 min, flow = 1 ml/min, 100% solvent A; t = 162 min, flow = 1 ml/min, 100% solvent A; t = 163 min, flow = 1 ml/min, 20% solvent A; t = 177 min,
flow = 1 ml/min, 20% solvent A; t = 178.5 min, flow = 0.4 ml/min, 20% solvent A. Total run time = 180 min. Samples were
injected in 80% acetonitrile (29).
Reversed phase HPLC was performed using a 4.6 × 150 mm, 3-µm
Hypersil ODS C18 column (Phenominex, Macclesfield, Cheshire, UK).
Solvent A was 50 mM formic acid adjusted to pH 5 with
triethylamine. Solvent B was 50% solvent A, 50% acetonitrile. Column
temperature was set to 30 °C. Gradient conditions were as follows:
t = 0 min, flow = 0.5 ml/min, 95% solvent A;
t = 30 min, flow = 0.5 ml/min, 95% solvent A;
t = 160 min, flow = 0.5 ml/min, 85% solvent A; t = 165 min, flow = 0.5 ml/min, 76% solvent A;
t = 166 min, flow = 1.5 ml/min, 5% solvent A;
t = 172 min, flow = 1.5 ml/min, 5% solvent A;
t = 173 min, flow = 1.5 ml/min, 95% solvent A;
t = 178 min, flow = 1.5 ml/min, 95% solvent A;
t = 179 min, flow = 0.5 ml/min, 95% solvent A. Total run time = 180 min. Samples were injected in 100% aqueous solution.
LC-ESI MS--
A Waters CapLC system was interfaced with a QTOF
hybrid quadrapole time-of-flight mass spectrometer with electrospray
ionization in positive mode, fitted with a Z-spray ion source
(Micromass UK, Ltd., Wythenshawe, Manchester, UK). A 1 × 150 mm
microbore NP-HPLC column was packed with stationary phase material from a GlycoSepN column (Glyko). The same solvents were used as for standard
NP-HPLC with a gradient from 80 to 50% acetonitrile over 120 min at a
flow rate of 10 µl/min. Positive ion mass spectra of the sialylated
O-glycans were recorded under the following conditions:
source temperature 90 °C; desolvation temperature 150 °C;
desolvation gas flow 200 liters/h; nebulizer gas 40 liters/h capillary
voltage 3000 V; cone voltage 30 V; mass range 50-3500.
MALDI-TOF MS--
Positive ion MALDI-TOF mass spectra were
recorded with a Micromass TofSpec 2E reflectron-TOF mass spectrometer
(Micromass UK, Ltd., Wythenshawe, Manchester, UK) fitted with delayed
extraction and a nitrogen laser (337 nm). The acceleration voltage was
20 kV; the pulse voltage was 3200 V, and the delay for the delayed extraction ion source was 500 ns. Samples were prepared by mixing 0.5 µl of the aqueous glycan solution and 0.5 µl of a saturated solution of 2,5-dihydroxybenzoic acid on the stainless steel MALDI target plate and allowing the mixture to dry at room temperature. The
sample:matrix mixture was then recrystallized from ethanol (30).
Deglycosylation and Proteolytic Fragmentation of
Apo(a)--
Limited proteolysis of apo(a) was carried out by
incubation with thermolysin (from Bacillus
thermoproteolyticus, 40 units/mg, Sigma) at a mass ratio of enzyme
to substrate of 1:200 in 0.125 M Tris-HCl, 0.15 M NaCl, 10 mM CaCl2 (pH 7.8) for 30 min at 37 °C (15). The reaction was stopped by addition of the
polyacrylamide gel electrophoresis loading buffer. Deglycosylation
of Lp(a) was performed as follows: Lp(a) (10 µg) was incubated with
CPS (0.05 units) and/or O-glycosidase (5 milliunits, Roche
Molecular Biochemicals) in 100 µl of phosphate buffer (pH 7.3)
overnight at 37 °C.
Electrophoresis and Immunoblotting--
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was performed on 4-10%
acrylamide gradient slab minigels (Mini Protean II, Bio-Rad), using a
discontinuous buffer system (31). Before electrophoresis, samples
(50-200 ng of apo(a) protein) were combined with glycerol, bromphenol
blue, and EDTA to final concentrations of 2%, 0.01%, and 0.05 mM, respectively. Reduced samples were prepared by boiling
at 100 °C for 4 min in the presence of 20 mM
dithiothreitol and 2% sodium dodecyl sulfate. Proteins were then
electroblotted onto nitrocellulose and revealed by immunoblotting (15).
The uniform transfer of all material was confirmed by Coomassie
staining gels after electroblotting and by reversible staining of the
nitrocellulose sheets with Ponceau S solution. Fragments of apo(a) were
revealed using a peroxidase-conjugated polyclonal anti-apo(a) antibody
(32). The polyclonal antibody reacts strongly with all apo(a) fragments
generated either in vivo or in vitro (15, 31).
Visualisation was achieved by enhanced chemiluminescence detection
(ECL, Amersham Pharmacia Biotech).
Prediction of Potential Apo(a) O-Glycan Sites--
Potential
sites for O-linked glycosylation on apo(a) were predicted by
comparison of the amino acid sequence of the specified interkringle
linker domains with known O-glycosylated proteins listed in
the O-GLYCBASE and using the NetOglyc software
(33).
Assignment of Apo(a) N- and O-Glycan Structures by Exoglycosidase
Sequencing--
Human Lp(a) was isolated from fasted plasma and apo(a)
purified as described previously (12). Apo(a) was then subjected first
to automated hydrazinolysis to remove all N- and
O-linked glycans, and the released sugars were fluorescently
labeled and analyzed by NP-HPLC. Two predominant N-linked
glycans were detected that accounted for more than 85% of the material
eluting in positions of common N-linked glycans (Fig. 1).
The G.U. values of the two major peaks and the products resulting from
their digestion with the listed exoglycosidases indicated that they
were biantennary complex oligosaccharides present in either a mono-
(A2G2S1) or disialylated (A2G2S2) state (Fig.
1A). Treatment of the
N-glycans with ABS resulted in the loss of both peaks and
formation of a single peak with a G.U. value identical to the neutral
biantennary structure, A2G2 (Fig. 1B). In the presence of
both ABS and BTG, the terminal galactose residues were also removed
leading to the formation of the common core structure
GlcNAc2Man3GlcNAc2, which is also
abbreviated as A2G0 (Fig. 1C). Because the conditions employed for hydrazinolysis of N-glycans can lead to the
partial destruction of O-glycans, apo(a) was also subjected
to a milder manual hydrazinolysis method to maximize the recovery of
O-glycans. This procedure yielded several compounds that
were labeled with 2-AB and analyzed by HPLC. An NP-HPLC profile of the
recovered peaks is shown in Fig. 2. The
peaks labeled with asterisks (as well as a compound that
coeluted with peak 1) were also detected in hydrazinolysis
"blanks" and were subsequently found to be derived from
carbohydrates associated with the filter paper used in the glycan
purification method. The apo(a) O-glycans detected were all
of the core type 1 structure (Gal
Treatment of the O-glycan pool with ABS resulted in the loss
of the sialylated structures and a concomitant increase in the level of
the nonsialylated core type 1 structure, Gal Confirmation of Glycan Structural Assignments by Mass
Spectrometry--
We next confirmed the structural assignments made
for all of the apo(a) glycans by mass spectrometry. The sialylated
O-glycans were analyzed by (NP) LC-ESI MS (Fig.
4). The two peaks eluted in their
predicted order with the monosialylated structure eluting at 60 min and
the disialylated structure eluting at 82 min (Fig. 4A). The
corresponding mass spectra for these peaks revealed molecular ions at
m/z values of 795.3 (Fig. 4B) and 1086.4 (Fig.
4C), respectively. These values represent the [M + H]+ ions for each glycan and are consistent with the
structural assignments made on the basis of the exoglycosidase
digestions and G.U. value determinations above. The mass spectrum for
NeuNAc
The structures of apo(a) glycans have not been determined previously;
however, predictions based on apo(a) monosaccharide composition have
been made (18, 34). A comparison of the published monosaccharide
composition of apo(a) compared with the predicted monosaccharide
composition based on our analysis of intact N- and
O-glycans is given in Table
III. The values for the three studies listed in Table III are in close agreement and indicate a mean apo(a)
monosaccharide molar ratio of 3:7:5:4:7 for
Man:Gal:GalNAc:GlcNAc:NeuNAc, respectively. However, the previous
assertion that the predominant apo(a) O-glycan structures
are Gal-GalNAc and NeuNAc-(NeuNAc-Gal)-GalNAc (18) appear to be
incorrect as our glycan sequencing and mass spectrometry data indicate
that NeuNAc-Gal-GalNAc is the most abundant O-glycan (Fig.
2). Comparison of the amounts of apo(a) N- and
O-glycans recovered from apo(a) revealed that they were present at a molar ratio of 1:5 (N-/O-glycans)
consistent with the previously published monosaccharide data (data not
shown).
Role of Apo(a) O-Glycans in Conferring Resistance to
Thermolysin Digestion--
Since apo(a) O-glycans are
present in the interkringle linker domains (20), we speculated that
they may protect potential protease-sensitive regions from enzyme
action. In order to test this hypothesis, we pretreated Lp(a) with CPS
or CPS plus O-glycosidase (which removes core Gal-GalNAc
from Ser and Thr residues) and assessed apo(a) susceptibility to
limited thermolysin digestion. In the presence of thermolysin, apo(a)
was degraded to form two peptides (Fig.
6A, lane 2) that have been
characterized previously and found to be the result of a cleavage
between Ala3532 and Phe3533 of the linker 4 domain that links kringle IV4 and kringle IV5 (12). When Lp(a) was
first treated with CPS to remove terminal sialic acids, two dominant
peptides were again formed after thermolysin treatment, and their
molecular weights were decreased compared with the peptides generated
without CPS pretreatment (Fig. 6A, compare lanes
2 and 3). This shift in molecular weight is
consistent with the loss of approximately 16-20 residues of
NeuNAc from each peptide. Interestingly, the larger N-terminal fragment
was also more degraded as evidenced by the smearing pattern below the
major band (Fig. 6A, lane 3). This suggests that
desialylation alone may alter the conformation of the interkringle
linkers to such an extent that thermolysin action is enhanced or that
the charge associated with the sialic acids normally repels access of
the apo(a) polypeptide to thermolysin. Evidence for lower molecular weight peptides was also seen below the C-terminal fragment, again consistent with a conformational change that favors thermolysin action
(Fig. 6A, lane 3). When Lp(a) was pretreated with CPS and O-glycosidase and then incubated with thermolysin, the vast
majority of the N-terminal peptide was degraded with less than 5% of
the material remaining in its original position (Fig. 6A, lane
4). The C-terminal peptide was also further degraded, although a
band remained with a molecular mass ~15 kDa less than the
original C-terminal peptide (Fig. 6A, lane 4). This may be
partially due to the loss of the remaining Gal-GalNAc residues or to
the formation of deglycosylated fragments of the original N-terminal
peptide.
The pretreatment of apo(a) with the O-glycosidase (which
specifically cleaves Gal-GalNAc) alone did not alter apo(a)
susceptibility to thermolysin digestion nor did it result in a change
in molecular weight of the two thermolysin proteolytic products (Fig.
6A, compare lanes 2 and 5). This final
result indicates that only a very small proportion of the apo(a)
glycans was present as Gal-GalNAc and that their removal does not make
a significant impact on the susceptibility of apo(a) to thermolysin
digestion. The fact that the molecular weight of the initial N-terminal
and C-terminal peptides remains constant after O-glycosidase
treatment also supports our oligosaccharide sequencing and mass
spectrometry data (Fig. 3 and Table II) showing that the predominant
O-glycan present is NeuNAc-Gal-GalNAc (which is not a
substrate for O-glycosidase).
Fig. 6B shows an additional Western blot from a separate
experiment. In this case the gel was intentionally overloaded in order
to detect any minor apo(a) fragments generated under the different
digestion conditions. The data are in close agreement with those shown
in Fig. 6A and confirm that desialylation of apo(a) results
in a partial increase in its sensitivity to thermolysin digestion, whereas complete removal of apo(a) O-glycans
dramatically increases its subsequent fragmentation.
Prediction of O-Glycosylation Potential of Interkringle Linker
Domains--
Previous work has revealed a
thermolysin-sensitive site for apo(a) cleavage in the kringle IV4
linker domain, i.e. between kringles 4 and 5 (12). Our
present data suggest that O-glycans present in the
interkringle linker domains may stabilize apo(a) by preventing
thermolysin proteolytic action. By using a computer algorithm that
compares polypeptide sequences with those of glycoproteins known to
contain O-glycans (33), we compared the
O-glycosylation potential for the interkringle linker
domains IV2, IV4, and IV7. In agreement with our hypothesis that
O-glycosylation can modulate apo(a) protease resistance, the
kringle IV4 linker was predicted to have no sites occupied by
O-glycans (Fig. 7). In
contrast, the kringle IV2 and IV7 linkers (as well as the other kringle IV linkers, data not shown) were predicted to be highly glycosylated (Fig. 7).
The present studies utilized exoglycosidase sequencing
techniques and mass spectrometry to determine the complete structure (including glycosidic linkage analysis) of human apo(a)
oligosaccharides. The predicted monosaccharide composition was in
general agreement with data published previously (18, 34). However, the
disialylated and non-sialylated O-glycan structures that
were predicted previously to occur (18) were found to account for only
10-20% of the apo(a) O-glycan pool (Fig. 3). The most
abundant oligosaccharide was, in fact, a monosialylated core type 1 O-glycan (Fig. 5), a structure that can also be deduced from
the previous monosaccharide analysis (18, 34).
The slightly lower ratio of NeuNAc:Gal observed in our study is due to
the finding that almost half of the biantennary N-glycans (which accounted for ~17% of total apo(a) glycans) were present in a
monosialylated state. In earlier studies (18), NeuNAc and Gal were
present at equimolar ratios, suggesting an absence of monosialylated
N-glycans. Possible reasons for this discrepancy may be
related to the high variation in sialic acid concentrations reported
(as determined by gas chromatography) or due to the nonspecificity of
the thiobarbituric acid method used in the NeuNAc quantitation (18).
Use of the thiobarbituric acid or "Warren" method can lead to an
overestimation of lipoprotein sialic acid levels (35, 36). It is
unlikely that the apo(a) N-glycans were desialylated during
processing in the present work as the O-glycan pool (which also contained NeuNAc in the When apo(a) was treated with O-glycosidase (which cleaves
Gal-GalNAc but not NeuNAc-Gal-GalNAc from Thr/Ser) followed by
thermolysin, there was no significant change in the molecular weight of
the resulting peptides compared with the peptides resulting from
treatment of apo(a) with thermolysin alone. This supports our
exoglycosidase sequencing data indicating that the Gal-GalNAc content
of the original apo(a) samples was low.
Having established the apo(a) oligosaccharide structures, we went on to
investigate the potential influence of the O-glycans on
apo(a) sensitivity to protease digestion. It is well known that human
plasma and urine contains apo(a) fragments (31, 37). The factors that
control the formation of these fragments are, however, poorly
understood. It appears that specific domains of apo(a) are more
susceptible to proteolytic cleavage than others (12). For example, the
major cut site for both thermolysin and neutrophil elastase (both
serine proteases) is in the kringle IV4 linker (12, 13). This domain is
predicted to be devoid of glycan chains (Fig. 7B), and we
hypothesized that the removal of O-glycans from other
linkers may result in increased susceptibility to protease digestion.
This was shown to be the case when O-glycans were trimmed by
sialidase treatment, and particularly when the entire oligosaccharide
structures were removed by treatment with sialidase and
O-glycosidase. The role of O-glycans in
protecting apo(a) from protease digestion may also explain why the
apo(a) peptide generated by cleavage at the elastase cut site in the kringle IV7 linker requires an extended incubation time or increase in
enzyme concentration (13). Other examples of O-glycans
conferring protease resistance have also been reported (see Ref. 17).
Interestingly, the Drosophila melanogaster "mucin-D"
glycoprotein contains a large amount (40% by mass) of Gal It is likely that apo(a) O- (and N-) glycans play
additional functional roles, for example in intracellular processing
(39, 40), maintaining the tertiary structure of apo(a), and preventing aggregation (17). Because apo(a) O-glycans are hydrophilic, they may also play a role in ensuring that the bulk of apo(a) is
extended out into the aqueous phase, as has been observed in structural
studies of recombinant Lp(a) (41).
Megalin/gp 330 has recently been identified as an endocytic receptor
for Lp(a) (42). This receptor also appears to be involved in the
cellular uptake of other glycosylated apolipoproteins including apoE,
apoJ, apoH, and apoB100 (43-46). Since megalin is highly expressed in
the brush border of renal proximal tubules and in the coated pits of
glomerular epithelial cells (47, 48), it provides a plausible control
mechanism for the generation of specific urinary apolipoproteins (49).
Interestingly, plasminogen is also a ligand for megalin (50), yet its
urinary excretion is extremely low compared with apo(a) (49). Since the
kringle type IV and type V structures of apo(a) are homologous to
plasminogen (2), it is tempting to speculate that the higher excretion of apo(a) may be related to the almost 14-fold higher content of
carbohydrate associated with the apo(a) kringle linkers. This possibility has not been addressed as far as we are aware.
The presence of terminal Gal residues on apo(a) may also confer an
ability to bind to the macrophage asialoglycoprotein receptor. An
analogous pathway has been proposed for apoB100 (LDL) endocytosis by
macrophages (51, 52). Arguing against such a pathway, one study has
shown that the removal of sialic acid residues from recombinant apo(a)
did not alter its binding to a partially characterized macrophage foam
cell surface receptor (53). Investigation of the full range of
functions for apo(a) oligosaccharides will clearly require further study.
The generation of apo(a) fragments in vivo has been
suggested to be potentially atherogenic due to the possibility of
C-terminal peptides interfering with fibrinolysis (15, 19). Of
potential relevance, apo(a) fragments accumulate in atherosclerotic
lesions where they may promote thrombogenesis (14). In the
microenvironment of the artery wall, it is possible that
macrophage-derived glycosidases and proteases act in concert to degrade
apo(a). This might have the unfortunate consequence of generating
macromolecular complexes between "mini-Lp(a)" and extracellular
matrix components that could then be taken up by macrophages to form
foam cells and thereby promote the development of the atherosclerotic
lesion. Since macrophages can release proteases (54, 55) and
glycosidases (56), our discovery that apo(a) glycans normally limit the
extent of proteolytic fragmentation might explain why, in the
macrophage-rich atherosclerotic lesion (57), apo(a) fragments are found
to accumulate (14). It would be interesting to assess the degree of
glycosylation present on such lesion-derived apo(a) fragments.
In conclusion, the present study has directly elucidated the structure
of human apo(a) oligosaccharide chains and shown that the
O-glycans play an important role in maintaining the
stability of apo(a) by conferring resistance to degradation by the
serine protease, thermolysin.
2-3Gal
1-3GalNAc, with smaller amounts of disialylated
and non-sialylated O-glycans also detected. Removal of
apo(a) O-glycans by sialidase and O-glycosidase
treatment dramatically increased the sensitivity of the polypeptide to
thermolysin digestion. These studies provide the first direct
sequencing data for apo(a) glycans and indicate a novel function for
apo(a) O-glycans that is potentially related to the
atherogenicity of Lp(a).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subject data and plasma lipids (mg/dl)
-elimination reaction
occurring at the glycosidic linkage between the core GalNAc and
sialylated Gal residues, a recognized characteristic of
O-glycan instability also known as "peeling" (23, 24), milder conditions of 60 °C for 6 h were manually employed (see below). The recovered glycan solutions were evaporated to dryness using
a vacuum centrifuge, and their reducing termini were fluorescently labeled by reductive amination with 2-aminobenzamide (2-AB) (25) using
a LudgerTag kit (Ludger, Oxford, UK).
1 to +2 cm from origin) by washing with
water. A rotary evaporator was used to concentrate samples before 2-AB
labeling and analysis by HPLC and mass spectrometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3GalNAc
1-R) and were found to
be present in different sialylated states. The predominant O-glycan detected was a monosialylated trisaccharide (peak
3) that was closely related to the disialylated (peak 4) and
nonsialylated (peak 1) structures that were also present (Fig.
2A). Peak 2 accounted for ~4% of the O-glycan
pool and was identified as NeuNAc
2-3Gal which is a degradation
product caused via a
-elimination reaction occurring at the
glycosidic linkage between the core GalNAc and sialylated Gal residues,
a recognized characteristic of O-glycan instability also
known as "peeling" (23, 24).
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Fig. 1.
Normal phase HPLC profiles of apo(a)
N-linked oligosaccharides and their exoglycosidase
digestion products. Human apo(a) was subjected to hydrazinolysis,
and the released glycans were labeled with 2-AB and analyzed by NP-HPLC
(A). The glycan pool was also treated with ABS
(B), and ABS + BTG (C), in order to sequence the
oligosaccharide chains (see "Experimental Procedures" for details).
The digestion of the two major complex biantennary structures
(peaks 1 and 2) to form the predicted truncated
products are indicated by the arrows. A2G2S2, disialylated
complex biantennary glycan containing 2 galactose residues; A2G2S1,
monosialylated complex biantennary glycan containing 2 galactose
residues; A2G2, nonsialylated complex biantennary glycan containing 2 galactose residues; A2G0, nonsialylated complex biantennary glycan
containing no galactose. G.U. values were derived from a dextran ladder
(29). The glycan structures are represented by the following symbols:
, mannose;
, N-acetylglucosamine;
, galactose;
,
N-acetylgalactosamine;
, N-acetylneuraminic
acid.
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Fig. 2.
Normal phase HPLC profile of apo(a)
O-linked oligosaccharides. Apo(a) samples were
subjected to a manual hydrazinolysis procedure at 60 °C for 6 h
and the recovered glycans 2-AB labeled and analyzed by NP-HPLC. The
structures illustrated were sequenced by exoglycosidase digestions (as
indicated in the legend to Fig. 1) and by comparison of the product and
precursor G.U. values with those of known standards. Conversion of the
sialylated structures to the neutral Gal-GalNAc product after ABS
treatment is indicated by the arrows. The glycan structures
are represented by the symbols described in the legend to Fig. 1. See
main text for further details.
1-3GalNAc (Fig.
2B). In the presence of ABS and BTG the Gal
1-3GalNAc
peak was partially removed, and the remaining non-digestible material appeared to be due to a contaminant introduced during the
hydrazinolysis procedure. In order to achieve an accurate determination
of the amount of Gal
1-3GalNAc normally present on apo(a), the
glycan mixture was also run on RP-HPLC to separate the contaminant from Gal
1-3GalNAc. The RP-HPLC profiles are shown in Fig.
3. The compounds labeled with
asterisks again represent peaks that were present in the
hydrazinolysis "blank," and the remaining peaks are numbered using
the same nomenclature as shown in Fig. 2A. The
Gal
1-3GalNAc (Fig. 3A, peak 1) was clearly separated
from the other compounds and was found to account for 11% of the
O-glycan pool. Note that the order of elution of peaks 2 and
3 changed under the RP-HPLC conditions as compared with the NP-HPLC
conditions (Figs. 3A and 2A, respectively). The
identity of peak 1 was confirmed by ABS digestion of the sialylated
structures (peaks 3 and 4) that were quantitatively recovered as
Gal
1-3GalNAc on the RP-HPLC system (Fig. 3B).
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Fig. 3.
Reversed phase HPLC profile of apo(a)
O-linked oligosaccharides. Apo(a) samples were
subjected to a manual hydrazinolysis procedure at 60 °C for 6 h
and the recovered glycans 2-AB labeled and analyzed by RP-HPLC. The
peaks were assigned according to the numbering scheme for the NP-HPLC
analysis (Fig. 2). Identity of the structures was confirmed by
digestion with ABS and by comparison of the product and precursor
arabinose unit (A.U.) values with those of known standards (29). See
main text for further details.
2-3Gal
1-3GalNAc also revealed ions at m/z
values of 817.3 and 839.3 that were due to the [M + Na]+
and [M
H + 2Na]+ (Na salt) ions, respectively
(Fig. 5). In the three apo(a) samples analyzed, NeuNAc
2-3Gal
1-3GalNAc was by far the most abundant glycan present accounting for >70% of the total glycan pool. The molecular masses for all of the N- and O-glycans
were also determined by analyzing the glycan pool by MALDI-TOF MS, and
the masses of the most abundant ions detected are given in Table
II along with their corresponding
glycan structures.
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Fig. 4.
Analysis of sialylated apo(a)
O-glycans by LC-ESI MS. Apo(a)
O-glycans were released by hydrazinolysis, 2-AB-labeled, and
analyzed by microbore NP-HPLC with in-line electrospray ionization
(+ve) mass spectrometry. A, chromatogram for 2-AB
labeled glycans with 330 nm Ex/440 nm Em fluorescence detection;
B, chromatogram for the +ve ion at m/z 795;
C, chromatogram for the +ve ion at m/z 1086. The
y ordinates show relative signal intensity.
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Fig. 5.
Mass spectrum (molecular ion region) of the
predominant apo(a) O-glycan. The mass spectrum
for the most abundant apo(a) O-glycan is shown after its
elution from the microbore HPLC column as illustrated in Fig.
4B. The three ions present were due to the [M + H]+ (m/z = 795.3), [M + Na]+
(m/z = 817.3), [M H + 2Na]+
(m/z = 839.3) ions of
NeuNAc
2-3GalNAc
1-3Gal-(2-AB).
Structure of apo(a) N-linked and O-linked glycans as 2-AB derivatives
Predicted monosaccharide composition of apo(a) compared to literature
values
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Fig. 6.
Influence of O-glycans on
apo(a) susceptibility to thermolysin digestion. Purified Lp(a) was
analyzed by Western blotting without enzymatic treatment (lane
1) or after partial thermolysin digestion in a fully glycosylated
state (lane 2) or after an initial treatment with CPS to
remove terminal sialic acids (lane 3) or CPS + O-glycosidase to remove sialylated core type 1 structures
(lane 4). A and B represent Western
blots from two independent experiments. A, Lp(a) was also
pretreated with O-glycosidase to remove non-sialylated core
type 1 (Gal-GalNAc-R) structures prior to thermolysin digestion
(A, lane 5). B, the gels were overloaded in order
to detect the quantitatively minor apo(a) fragments. The presence of
naturally occurring apo(a) fragments associated with the non-digested
Lp(a) is also evident in the overloaded condition (B, lane
1). Enzymatic deglycosylation of Lp(a) without thermolysin
digestion did not reduce antibody binding to apo(a) (not shown).
Results are representative of three independent experiments. See
"Experimental Procedures" for further details.
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Fig. 7.
Potential sites for apo(a)
O-linked glycosylation. Potential sites for
O-linked glycosylation on the linker domains between kringle
IV2-(IV2)IV3 (A), kringle IV4-IV5 (B), and
kringle IV7-IV8 (C) were predicted by amino acid sequence
comparison with known O-glycosylated proteins listed in
the O-GLYCBASE and using the NetOglyc software. The amino
acid sequences for each of the kringle linkers is given on the
x ordinate, and the potential for glycosylation is indicated
on the y ordinate. The horizontal line on each
panel represents the threshold value for glycosylation (i.e.
if the bars on the histogram are higher than the threshold
value, O-glycosylation is predicted to occur).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-3 linkage to Gal) did not contain a
predominant nonsialylated structure. We have also observed that human
apoB100 contains a high proportion of its N-glycans as a monosialylated complex biantennary structure, and this was noted when
the glycans were removed from apoB100 by either hydrazinolysis or
treatment with peptide N-glycosidase
F,2 indicating that under our
hydrazinolysis conditions the NeuNAc
2-3Gal glycosidic linkage is stable.
1-3GalNAc
that renders it highly resistant to protease action (38), consistent
with our data showing that the same disaccharide alone (generated after
treatment of Lp(a) with sialidase) provided resistance to thermolysin digestion.
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ACKNOWLEDGEMENTS |
---|
We thank Chantal Doucet (INSERM U551) for technical assistance. We also thank Dr. Michael Frischmann for providing the pure apo(a) from subject 3 and Prof. Raymond Dwek for helpful comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by a Wellcome Trust fellowship and Grant 058833 (to B. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Oxford Glycobiology Institute, Dept. of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, UK. Tel.: 44-1865-275780; Fax: 44-1865-275216; E-mail: brett@glycob.ox.ac.uk.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M102150200
2 B. Garner, D. J. Harvey, M. Frischmann, F. Nigon, M. J. Chapman, and P. M. Rudd, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a); 2-AB, 2-aminobenzamide; ABS, A. ureafaciens sialidase; BTG, bovine testes galactosidase; CPS, C. perfringens sialidase; NP-HPLC, normal phase high performance liquid chromatography; RP-HPLC, reversed phase high performance liquid chromatography; LC-ESI MS, liquid chromatography-electrospray ionization mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; G.U., glucose units; A.U., arabinose units; A2G2S2, disialylated complex biantennary glycan containing 2 galactose residues; A2G2S1, monosialylated complex biantennary glycan containing 2 galactose residues; A2G2, nonsialylated complex biantennary glycan containing 2 galactose residues; A2G0, nonsialylated complex biantennary glycan containing no galactose; LDL, low density lipoprotein.
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REFERENCES |
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---|
1. | Brunner, C., Kraft, H. G., Utermann, G., and Muller, H. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11643-11647[Abstract] |
2. | McLean, J. W., Tomlinson, J. E., Kuang, W. J., Eaton, D. L., Chen, E. Y., Fless, G. M., Scanu, A. M., and Lawn, R. M. (1987) Nature 330, 132-137[CrossRef][Medline] [Order article via Infotrieve] |
3. | Harpel, P. C., Gordon, B. R., and Parker, T. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3847-3851[Abstract] |
4. | Kronenberg, F., Steinmetz, A., Kostner, G. M., and Dieplinger, H. (1996) Crit. Rev. Clin. Lab. Sci. 33, 495-543[Medline] [Order article via Infotrieve] |
5. | Edelstein, C., Italia, J. A., Klezovitch, O., and Scanu, A. M. (1996) J. Lipid Res. 37, 1786-1801[Abstract] |
6. |
Klezovitch, O.,
Edelstein, C.,
Zhu, L.,
and Scanu, A. M.
(1998)
J. Biol. Chem.
273,
23856-23865 |
7. |
Etingin, O. R.,
Hajjar, D. P.,
Hajjar, K. A.,
Harpel, P. C.,
and Nachman, R. L.
(1991)
J. Biol. Chem.
266,
2459-2465 |
8. | Hajjar, K. A., Gavish, D., Breslow, J. L., and Nachman, R. L. (1989) Nature 339, 303-305[CrossRef][Medline] [Order article via Infotrieve] |
9. | Miles, L. A., Fless, G. M., Levin, E. G., Scanu, A. M., and Plow, E. F. (1989) Nature 339, 301-303[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Mooser, V.,
Marcovina, S. M.,
White, A. L.,
and Hobbs, H. H.
(1996)
J. Clin. Invest.
98,
2414-2424 |
11. |
Mooser, V.,
Seabra, M. C.,
Abedin, M.,
Landschulz, K. T.,
Marcovina, S.,
and Hobbs, H. H.
(1996)
J. Clin. Invest.
97,
858-864 |
12. | Huby, T., Schroder, W., Doucet, C., Chapman, J., and Thillet, J. (1995) Biochemistry 34, 7385-7393[Medline] [Order article via Infotrieve] |
13. |
Edelstein, C.,
Italia, J. A.,
and Scanu, A. M.
(1997)
J. Biol. Chem.
272,
11079-11087 |
14. | Hoff, H. F., O'Neil, J., Smejkal, G. B., and Yashiro, A. (1994) Chem. Phys. Lipids 67-68, 271-280 |
15. | Huby, T., Doucet, C., Dieplinger, H., Chapman, J., and Thillet, J. (1994) Biochemistry 33, 3335-3341[Medline] [Order article via Infotrieve] |
16. |
Edelstein, C.,
Shapiro, S. D.,
Klezovitch, O.,
and Scanu, A. M.
(1999)
J. Biol. Chem.
274,
10019-10023 |
17. | Van den Steen, P., Rudd, P. M., Dwek, R. A., and Opdenakker, G. (1998) Crit. Rev. Biochem. Mol. Biol. 33, 151-208[Abstract] |
18. |
Fless, G. M.,
ZumMallen, M. E.,
and Scanu, A. M.
(1986)
J. Biol. Chem.
261,
8712-8718 |
19. | Scanu, A. M., and Edelstein, C. (1997) J. Lipid Res. 38, 2193-2206[Abstract] |
20. | Kratzin, H., Armstrong, V. W., Niehaus, M., Hilschmann, N., and Seidel, D. (1987) Biol. Chem. Hoppe Seyler 368, 1533-1544[Medline] [Order article via Infotrieve] |
21. | Doucet, C., Huby, T., Chapman, J., and Thillet, J. (1994) J. Lipid Res. 35, 263-270[Abstract] |
22. | Merry, A. H., Bruce, J., Bigge, C., and Ioannides, A. (1992) Biochem. Soc. Trans. 20, 91 |
23. | Chamow, S. M., and Hedrick, J. L. (1988) Carbohydr. Res. 176, 195-203[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Kanamori, A.,
Inoue, S.,
Iwasaki, M.,
Kitajima, K.,
Kawai, G.,
Yokoyama, S.,
and Inoue, Y.
(1990)
J. Biol. Chem.
265,
21811-21819 |
25. | Bigge, J. C., Patel, T. P., Bruce, J. A., Goulding, P. N., Charles, S. M., and Parekh, R. B. (1995) Anal. Biochem. 230, 229-238[CrossRef][Medline] [Order article via Infotrieve] |
26. | Ashford, D., Dwek, R. A., Welply, J. K., Amatayakul, S., Homans, S. W., Lis, H., Taylor, G. N., Sharon, N., and Rademacher, T. W. (1987) Eur. J. Biochem. 166, 311-320[Abstract] |
27. | Mattu, T. S., Royle, L., Langridge, J., Wormald, M. R., Van den Steen, P. E., Van Damme, J., Opdenakker, G., Harvey, D. J., Dwek, R. A., and Rudd, P. M. (2000) Biochemistry 39, 15695-15704[CrossRef][Medline] [Order article via Infotrieve] |
28. | Rudd, P. M., Guile, G. R., Kuster, B., Harvey, D. J., Opdenakker, G., and Dwek, R. A. (1997) Nature 388, 205-207[CrossRef][Medline] [Order article via Infotrieve] |
29. | Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B., and Dwek, R. A. (1996) Anal. Biochem. 240, 210-226[CrossRef][Medline] [Order article via Infotrieve] |
30. | Harvey, D. J. (1993) Rapid Commun. Mass Spectrom. 7, 614-619[Medline] [Order article via Infotrieve] |
31. |
Doucet, C.,
Mooser, V.,
Gonbert, S.,
Raymond, F.,
Chapman, J.,
Jacobs, C.,
and Thillet, J.
(2000)
J. Am. Soc. Nephrol.
11,
507-513 |
32. | Guo, H. C., Armstrong, V. W., Luc, G., Billardon, C., Goulinet, S., Nustede, R., Seidel, D., and Chapman, M. J. (1989) J. Lipid Res. 30, 23-37[Abstract] |
33. | Hansen, J. E., Lund, O., Tolstrup, N., Gooley, A. A., Williams, K. L., and Brunak, S. (1998) Glycoconj. J. 15, 115-130[CrossRef][Medline] [Order article via Infotrieve] |
34. | Seman, L. J., and Breckenridge, W. C. (1986) Biochem. Cell Biol. 64, 999-1009[Medline] [Order article via Infotrieve] |
35. |
Warren, L.
(1959)
J. Biol. Chem.
234,
1971-1975 |
36. |
Sobenin, I. A.,
Tertov, V. V.,
and Orekhov, A. N.
(1998)
J. Lipid Res.
39,
2293-2299 |
37. | Oida, K., Takai, H., Maeda, H., Takahashi, S., Shimada, A., Suzuki, J., Tamai, T., Nakai, T., and Miyabo, S. (1992) Clin. Chem. 38, 2244-2248[Abstract] |
38. | Kramerov, A. A., Arbatsky, N. P., Rozovsky, Y. M., Mikhaleva, E. A., Polesskaya, O. O., Gvozdev, V. A., and Shibaev, V. N. (1996) FEBS Lett. 378, 213-218[CrossRef][Medline] [Order article via Infotrieve] |
39. |
White, A. L.,
Guerra, B.,
Wang, J.,
and Lanford, R. E.
(1999)
J. Lipid Res.
40,
275-286 |
40. | Wang, J., and White, A. L. (1999) Biochem. Soc. Trans. 27, 453-458[Medline] [Order article via Infotrieve] |
41. | Phillips, M. L., Lembertas, A. V., Schumaker, V. N., Lawn, R. M., Shire, S. J., and Zioncheck, T. F. (1993) Biochemistry 32, 3722-3728[Medline] [Order article via Infotrieve] |
42. |
Niemeier, A.,
Willnow, T.,
Dieplinger, H.,
Jacobsen, C.,
Meyer, N.,
Hilpert, J.,
and Beisiegel, U.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
552-561 |
43. |
Willnow, T. E.,
Goldstein, J. L.,
Orth, K.,
Brown, M. S.,
and Herz, J.
(1992)
J. Biol. Chem.
267,
26172-26180 |
44. |
Kounnas, M. Z.,
Loukinova, E. B.,
Stefansson, S.,
Harmony, J. A.,
Brewer, B. H.,
Strickland, D. K.,
and Argraves, W. S.
(1995)
J. Biol. Chem.
270,
13070-13075 |
45. |
Moestrup, S. K.,
Schousboe, I.,
Jacobsen, C.,
Leheste, J. R.,
Christensen, E. I.,
and Willnow, T. E.
(1998)
J. Clin. Invest.
102,
902-909 |
46. |
Stefansson, S.,
Chappell, D. A.,
Argraves, K. M.,
Strickland, D. K.,
and Argraves, W. S.
(1995)
J. Biol. Chem.
270,
19417-19421 |
47. |
Kanalas, J. J.,
and Makker, S. P.
(1988)
J. Immunol.
141,
4152-4157 |
48. | Kanalas, J. J., and Makker, S. P. (1990) J. Am. Soc. Nephrol. 1, 792-798[Abstract] |
49. | Kostner, K., Spitzauer, S., Rumpold, H., Maurer, G., Knipping, G., Hrzenjak, A., Frank, S., and Kostner, G. M. (2001) Clin. Chim. Acta 304, 29-37[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Kanalas, J. J.,
and Makker, S. P.
(1991)
J. Biol. Chem.
266,
10825-10829 |
51. | Bartlett, A. L., and Stanley, K. K. (1998) Atherosclerosis 138, 237-245[CrossRef][Medline] [Order article via Infotrieve] |
52. | Bartlett, A. L., Grewal, T., De Angelis, E., Myers, S., and Stanley, K. K. (2000) Atherosclerosis 153, 219-230[CrossRef][Medline] [Order article via Infotrieve] |
53. | Keesler, G. A., Li, Y., Skiba, P. J., Fless, G. M., and Tabas, I. (1994) Arterioscler. Thromb. 14, 1337-1345[Abstract] |
54. | Powers, J. C., Gupton, B. F., Harley, A. D., Nishino, N., and Whitley, R. J. (1977) Biochim. Biophys. Acta 485, 156-166[Medline] [Order article via Infotrieve] |
55. | Galis, Z. S., Muszynski, M., Sukhova, G. K., Simon-Morrissey, E., and Libby, P. (1995) Ann. N. Y. Acad. Sci. 748, 501-507[Abstract] |
56. | Bolton, E. J., Jessup, W., Stanley, K. K., and Dean, R. T. (1997) Biochim. Biophys. Acta 1356, 12-22[Medline] [Order article via Infotrieve] |
57. | Gerrity, R. G. (1981) Am. J. Pathol. 103, 181-190[Abstract] |