From the
Advanced glycosylation end products (AGEs) arise from
glucose-derived Amadori products and have been implicated in the
pathogenesis of diabetic vascular disease. We recently reported the
presence of an AGE-modified form of low density lipoprotein (LDL) that
circulates in high amounts in patients with diabetes or renal
insufficiency and that exhibits impaired plasma clearance kinetics. We
utilized AGE-specific antibodies to identify the major sites of AGE
modification within protease-digested preparations of apolipoprotein B
that impair the binding of the AGE-modified form of LDL by human
fibroblast LDL receptors. The predominant site of AGE immunoreactivity
was found to lie within a single, 67-amino acid region located 1791
residues NH
Patients with diabetes or renal insufficiency suffer a high
incidence of atherosclerotic vascular disease, which has been
attributed in part to persistent elevation in the plasma level of the
lipoprotein components very low density lipoprotein and LDL
We
recently described an AGE-modified form of low density lipoprotein
(AGE-LDL) that circulates in elevated amounts in patients with diabetes
or renal insufficiency
(12) . The AGE modification of LDL
interferes significantly with its normal, receptor-mediated uptake, as
shown by fractional clearance studies performed in transgenic mice
expressing the human LDL receptor
(13) . The excessive formation
of AGEs on LDL thus has been proposed to be an important mechanism for
the dyslipidemia and accelerated atherogenesis that often is observed
in patients with diabetes or renal insufficiency. To gain additional
insight into the mechanism by which advanced glycosylation interferes
with the uptake and degradation of LDL, we have investigated by peptide
mapping the precise site(s) of AGE modification of apolipoprotein B
(apoB). As a model reaction, native LDL first was modified by glucose
in vitro to achieve the level of AGE modification that is
present in vivo in the plasma of diabetic and renally impaired
individuals
(12, 13) . AGE-LDL prepared in this manner
showed diminished recognition and uptake by human fibroblast LDL
receptors. AGE-specific antibodies were then used to probe protease V8
digests of AGE-LDL for the presence of distinct, AGE-modified peptides.
These peptides were isolated, and their position within apoB was
established by amino acid sequencing.
As a model reaction for studying AGE-induced alterations in
the structural and functional properties of LDL, we prepared
AGE-modified LDL in vitro by incubating freshly isolated,
native LDL with glucose
(12) . The incubation of glucose (200
mM) with LDL (2.5 mg/ml) for 14 days, for example, produced a
level of modification of 80 units of AGE/mg of apoB, comparable with
that present in the circulating LDL of patients with diabetes and renal
insufficiency
(13) . Although the glucose concentrations used to
prepare AGE-LDL in vitro were supraphysiological, it is
important to note that in vivo AGE modification results not
only from glucose but also from more reactive AGE intermediates that
arise from the catabolism of AGE-modified proteins
(5, 6) .
To assess the LDL receptor binding activity
of AGE-modified LDL, we utilized a competitive ligand binding assay in
human fibroblasts that has been used previously for the identification
of mutant LDL species
(17) . Fig. 1shows that a
progressive increase in the degree of AGE modification of LDL is
associated with a progressively decreased ability of the LDL to be
recognized and taken up by LDL receptors. Although the human fibroblast
uptake assay is relatively insensitive to the oxidative modification of
LDL
(18) , the possibility that the effects of advanced
glycosylation on LDL binding were due to oxidative changes was
minimized by adding the antioxidant BHT to the LDL/glucose incubation
mixtures. AGE-LDL prepared in this manner showed no increase in lipid
oxidation products compared with the control, native LDL when assessed
by reactivity with thiobarbituric acid (data not shown).
To identify the sites on apoB that are modified
by advanced glycosylation, AGE-LDL (80 units of AGE/mg of apoB) was
subjected first to varying degrees of enzymatic hydrolysis with
trypsin, chymotrypsin, protease V8, or a combination of two of these
enzymes. The products were then analyzed by polyacrylamide gel
electrophoresis and Western blotting using AGE-specific antibodies.
Preliminary studies showed that of the various peptide patterns that
were generated, those produced by partial protease V8 hydrolysis showed
both the simplest digestion pattern and the highest electrophoretic
resolution after silver staining and Western blotting analyses
(Fig. 2). Significant immunoreactivity was localized to two
peptide bands with apparent molecular masses of 28 and 15 kDa. A
qualitatively similar pattern of immunoreactivity was observed in
peptide digests that were probed with a monoclonal anti-AGE antibody in
place of the polyclonal reagent. Anti-AGE antibody reactivity within
these peptides also was found to be diminished in LDL that had been
incubated with glucose in the presence of the advanced glycosylation
inhibitor aminoguanidine. In addition, all staining could be shown to
be specifically abolished by co-incubating the primary antibody with
AGE-modified bovine serum albumin (not shown). A low but detectable
level of AGE immunoreactivity was consistently found to be present in
native LDL (not modified with glucose in vitro), verifying
previous observations that measurable quantities of AGE-modified LDL
also circulate in normal, nondiabetic individuals
(12, 13) .
A variety of experimental studies have established
that the primary amino group of lysine (or of an
To provide further evidence
for the absence of significant, structural modifications within the LDL
receptor binding domain of AGE-LDL, we probed for differences in the
immunoreactivity of native versus AGE-modified LDL with a
panel of antibodies directed against epitopes located near the receptor
binding region (apoB
Finally, we have
begun to analyze the site(s) of apoB advanced glycosylation in LDL
isolated directly from diabetic patients. Both a 28- and a 15-kDa
peptide species were identified by Western blotting to be modified by
advanced glycosylation in diabetic LDL, and preliminary results
indicate that the NH
Chemical modification of basic residues within apoB has been
shown previously to interfere with the ability of LDL to undergo
receptor-mediated uptake and degradation
(25, 26) .
Furthermore, borohydride stabilization of slowly reversible Amadori
adducts ex vivo can impair the normal plasma clearance
properties of LDL modified by early glycation products
(27) .
Because AGE moieties, once formed, remain irreversibly bound to lysine
residues, we sought to identify the precise sites of apoB modification
by AGEs that are associated with decreased LDL receptor binding
activity. Despite the very large size of apoB (4536 amino acids) and
its high content of potentially reactive lysine residues (359 lysine
residues), the predominant site of AGE immunoreactivity mapped to a
single 67-amino acid region located 1791 residues
NH
The observation that AGE modification
predominantly affects only a single, small region within apoB points to
the high reactivity of this site toward advanced glycosylation. This
result was not completely unanticipated given the observation that LDL
accumulates a measurable quantity of AGE modification despite having an
average circulating half-life of only 36-48 h
(12, 13) . The precise reason for the high
susceptibility of this region of apoB to AGE formation is unknown. The
formation of the initial glucose-derived Schiff base, for example, is
known to be favored at sites of unprotonated, nucleophilic amino groups
(24) . Within this context, recent
Although the present data cannot exclude
the possibility that biologically important sites of AGE modification
also occur elsewhere within LDL, an important question posed by these
findings is by what mechanism can the modification of lysine residue(s)
remote from the receptor binding domain of apoB have such a profound
impact on LDL receptor binding. Presumably, the presence of the AGE
modification induces a conformational change that is sufficient to
perturb ligand-receptor interaction. This may be the result of a charge
alteration in the affected lysine or, conceivably, of the formation of
metastable AGE cross-links with more remote regions of the apoB
molecule. An emerging body of literature suggests that an elaborate
spacial geometry is involved in the binding of the LDL particle to its
receptor. The mutation responsible for the syndrome of familial
defective apolipoprotein B-100, for instance, results in a Glu
In conclusion, the present data provide a
structural basis for beginning to assess how advanced glycosylation can
exert such profound functional alterations in the clearance properties
of LDL. More detailed investigation into the reactivity of apoB toward
AGEs, changes in the three-dimensional structural changes induced in
LDL by AGE formation, and the precise chemical nature of the apoB AGE
should assist ultimately in the design of more specific pharmacological
inhibitors of LDL-advanced glycosylation.
We are grateful to Dr. J. Fernandez and to the
Rockefeller University Protein Sequencing Facility, which is supported
in part by National Institutes of Health shared instrumentation grants
and by funds provided by the U. S. Army and Navy for the purchase of
equipment.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal of the putative LDL receptor binding
domain. These data point to the high reactivity and specificity of this
site for AGE formation and provide further evidence for important
structural interactions between the LDL receptor binding domain and
remote regions of the apolipoprotein B polypeptide.
(
)(1, 2, 3, 4) . Diabetic or
renally impaired patients also exhibit high circulating levels of
protein- and lipid-bound advanced glycosylation end products (AGEs).
AGEs arise by a succession of nonenzymatic rearrangement reactions,
beginning with glucose-derived Amadori products and proceeding to the
formation of highly reactive species capable of covalently modifying
and irreversibly cross-linking protein amino groups. Although
circulating AGE proteins form in part by the reaction of glucose with
serum proteins in situ, recent data suggest that a large
portion of the AGEs present in the blood compartment also arises from
the catabolism of AGE-modified tissue proteins
(5, 6) .
Thus, high circulating levels of reactive, AGE peptides can occur under
normoglycemic conditions if plasma filtration is impaired by mild to
moderate renal insufficiency. Although the precise structure(s) of the
major AGEs that form in vivo remains to be established,
advanced glycosylation has been found to affect a number of proteins,
and a large body of literature has implicated either enhanced AGE
formation or defective AGE clearance in the pathogenesis of several
age- and diabetes-related sequelae
(7, 8, 9, 10, 11) .
Preparation of AGE-LDL
Plasma LDL ( d = 1.025-1.063) was isolated from control
(nondiabetic, normal renal function) plasma by ultracentrifugation
(14) . AGE-modified LDL was prepared by incubating LDL (2.5 mg)
with glucose (200 mM) at 37 °C for 4-14 days in
phosphate-buffered saline containing 1 mM EDTA and 20
µM BHT. BHT was added to prevent AGE-mediated oxidative
changes to LDL, which arise as a consequence of phospholipid advanced
glycosylation
(12) . AGE-LDL prepared under these conditions
showed insignificant amounts of lipid oxidation when analyzed for
reactivity with thiobarbituric acid
(15) . To inhibit AGE
formation, aminoguanidine was added to selected incubations to a final
concentration of 300 mM. The LDL was then dialyzed against
phosphate-buffered saline containing 1 mM EDTA and 10
µM BHT, and aliquots were subjected to analysis by an
AGE-specific enzyme-linked immunosorbent assay
(12, 16) .
LDL Receptor Binding Studies
Lipoprotein uptake
and competition experiments were performed as described previously
(17) with cultured normal human foreskin fibroblasts. Native LDL
was iodinated with NaI, and the
I-labeled
LDL was added to cells to a final concentration of 2 µg of
protein/ml together with increasing amounts of various unlabeled LDLs.
After incubation for 2 h at 4 °C, surface bound radioactivity was
determined, and the percent bound was plotted as a function of added,
unlabeled competitor LDL
(17, 18) .
Peptide Mapping and Western Blotting
Analysis
Partial enzymatic hydrolysis was performed by adding 1
mg of native LDL (2 units of AGE/mg of apoB), AGE-modified LDL (80
units of AGE/mg of apoB), or AGE-modified LDL prepared in the presence
of aminoguanidine (20 units of AGE/mg of apoB) to increasing amounts of
trypsin, chymotrypsin, or Staphylococcus aureus protease V8
(5-50 µg, Boehringer Mannheim). The digestions were performed
in 0.2 M NaPO
buffer (pH 7.4) by overnight
incubation at 37 °C. The reactions were terminated by adding an
equal volume of CHCl
/MeOH, vortexing, and removing the
peptide-containing aqueous phase and interface. The peptides were
concentrated by vacuum centrifugation and redissolved in 2
Laemmli sample buffer, and the hydrolysis products were resolved by
electrophoresis in SDS-polyacrylamide gels (15-20% gradient).
Peptide products first were visualized by silver staining. Peptides
from duplicate samples that were electrophoresed in adjacent lanes were
then transferred onto nitrocellulose membranes
(19) and
incubated with a polyclonal anti-AGE antiserum (diluted 1:200), a
monoclonal anti-AGE IgG
(5 µg/ml, provided by Dr. H.
Founds, Alteon, Inc.), preimmune serum, or control IgG
antibody
(16) . The polyclonal anti-AGE antibody binds to
a major class of carbohydrate-derived AGEs that form in vivo and does not recognize acetyl- or malonyldialdehyde-modified LDL
(16) . AGE-containing bands were visualized by the addition of
an immunoperoxidase-labeled secondary antibody and development with
chloronaphthol/H
O
. To validate the specificity
of the anti-AGE antibodies, control experiments were performed in which
AGE-BSA was added to the primary antibody incubation at a final
concentration of 1 mg/ml. Complete blocking of AGE immunoreactivity was
observed under these conditions.
Amino Acid Sequencing
For NH-terminal
amino acid sequence analysis, the AGE-reactive peptides were
transferred to polyvinylidene difluoride membranes and subjected to
automated gas phase sequencing (Applied Biosystems, Foster City, CA)
(19) . For internal amino acid sequence analysis,
nitrocellulose-immobilized AGE-modified peptides were first digested
in situ with endoproteinase Lys-C
(20) . Cleavage
products were then eluted from the membrane, isolated by reverse-phase
HPLC employing a C-18 column and gradient elution with a
trifluoroacetic acid/H
O/acetonitrile buffer system
(21) , and microsequenced with an ABI gas phase sequencer.
LDL Epitope Analysis by Enzyme-linked Immunosorbent
Assay
Native and AGE-modified LDLs were analyzed for reactivity
to antibodies directed against synthetic apoB peptides whose sequences
are located in close proximity to the LDL receptor binding domain
(22) . 96-well microtiter plates were coated overnight with
either native LDL or AGE-LDL (10 µg/ml) and then incubated with
dilutions of the following anti-apoB peptide antibodies:
anti-apoB,
anti-apoB
,
anti-apoB
,
anti-apoB
, and
anti-apoB
. After washing, the bound
antibodies were detected with an alkaline phosphatase-linked secondary
antibody and p-nitrophenol as substrate.
Figure 1:
Inhibition of
LDL receptor binding of I-LDL by control, native LDL and
AGE-LDL. Upper panel, control LDL (
), AGE-LDL (80 units
of AGE/mg of apoB) (
), and AGE-LDL prepared in the presence of
aminoguanidine (20 units of AGE/mg of apoB) (
). Lower
panel, control LDL (
), AGE-LDL (9 units of AGE/mg of apoB)
(
), and AGE-LDL prepared in the presence of aminoguanidine (5
units of AGE/mg of apoB) (
). Advanced glycosylation reactions were
performed by incubating control LDL (2.5 mg/ml,
2 units of AGE/mg
of apoB) with glucose (200 mM for 14 ( upper panel) or
4 days ( lower panel)) in aminoguanidine (300 mM,
where indicated) in 0.2 M NaPO
buffer containing 1
mM EDTA and 20 µM BHT. Human foreskin fibroblasts
were incubated at 4 °C with
I-labeled control LDL
together with increasing amounts of unlabeled competitor LDL
preparations, and the
I-LDL binding was measured as
described under ``Materials and Methods.'' The data points
were calculated from the means of duplicate wells and displayed a
variation of <10%.
AGE-LDL
preparations modified to the level of 80 units of AGE/mg of apoB showed
virtually no binding to human fibroblast LDL receptors. By contrast,
LDL modified by glucose under the same incubation conditions (200
mM glucose for 14 days) except for the presence of the
advanced glycosylation inhibitor aminoguanidine (300 mM)
showed a lower level of AGE modification (20 units of AGE/mg of apoB)
and a correspondingly improved capacity to be bound by LDL receptors.
Even AGE-LDL displaying a low level of AGE modification (9 units of
AGE/mg of apoB, produced by incubating LDL with 200 mM glucose
for 4 days) exhibited impaired binding to fibroblast LDL receptors.
Furthermore, the receptor binding properties of this AGE-LDL were
readily distinguished from those of a corresponding preparation of LDL
modified by glucose in the presence of aminoguanidine (yielding 5 units
of AGE/mg of apoB).
Figure 2:
SDS-polyacrylamide gel electrophoresis and
Western blotting analyses of protease V8-digested preparations of
control, native LDL (2 units of AGE/mg of apoB), AGE-LDL (80 units
of AGE/mg of apoB), and AGE-LDL prepared in the presence of 300
mM aminoguanidine ( +AG) (20 units of AGE/mg of
apoB). Left panel, silver staining analysis. Right
panel, Western blotting analysis with a polyclonal anti-AGE
antibody. Also present but not well visualized in the photographic
reproduction are positively stained 28- and 15-kDa peptide bands in the
control, native LDL lane.
The AGE immunoreactive fragments were then
subjected to gas phase microsequencing to determine the precise
positions of the 28- and 15-kDa AGE-modified peptides within the apoB
polypeptide. As shown in Fig. 3, NH-terminal
microsequencing of the 28-kDa species identified that this peptide
begins at amino acid 1308 of the apoB sequence
(23) . Molecular
weight calculations of potential COOH-terminal cleavage sites predicted
a carboxyl terminus at amino acid 1536. The identity of this peptide
was validated by NH
-terminal sequencing of a second 28-kDa
AGE-modified peptide isolated from a different preparation of AGE-LDL
and by further internal digestion of the 28-kDa species with
endoproteinase Lys-C. Microsequence analysis of this second, smaller
peptide yielded an NH
terminus at position 1366 of the apoB
sequence.
Figure 3:
Summary of amino acid sequencing analyses
of the 28- and 15-kDa AGE-modified apoB peptides isolated after
protease digestion. (A) and (A`) refer to the
NH-terminal sequences obtained from two independently
isolated preparations of AGE-LDL. (B) refers to the
NH
-terminal sequence obtained after digestion in situ of the 28-kDa peptide with endoproteinase Lys-C. (C) is
the 15-kDa NH
-terminal sequence. Potential sites of lysine
modification are underlined.
NH-terminal microsequence analysis of the
15-kDa AGE-modified peptide gave the same amino terminus as that of the
28-kDa peptide, indicating that this fragment was derived from the
``parent'' 28-kDa peptide by an additional protease V8
cleavage. The carboxyl terminus of this peptide was estimated to lie at
amino acid 1455.
-amino acid)
initiates the advanced glycosylation process. Arginine and other
residues may then participate secondarily in AGE-mediated cross-linking
reactions
(24) . Examination of amino acid recoveries within the
NH
-terminal sequenced regions of the 28- and 15-kDa apoB
peptides revealed no quantitative loss of lysine residues or appearance
of novel amino acid peaks. Therefore, in all likelihood, these lysine
residues are not sites of advanced glycosylation within the
AGE-modified peptides. AGE modification thus appears to involve (at
least) 1 of 9 lysine residues (Lys
, Lys
,
Lys
, Lys
, Lys
,
Lys
, or Lys
) present within a
67-amino acid domain located between residues 1388 and 1454 of the apoB
primary sequence. Further attempts to localize AGE immunoreactivity to
even smaller peptide fragments by additional protease digestion
followed by gel electrophoresis or HPLC purification were frustrated by
an increase in the size heterogeneity and a corresponding decrease in
the yield of individual peptide species.
,
apoB
, apoB
,
apoB
, and apoB
)
(22) . No detectable differences in antibody reactivity were
detected when native and AGE-LDL were analyzed by an enzyme-linked
immunosorbent assay (not shown). These data do not exclude the
possibility that an immunochemically ``silent'' AGE or other
chemical modification occurs within the receptor binding domain of
AGE-modified LDL. Nevertheless, it provides additional support for the
lack of significant, structural alterations in regions that are in
close proximity to the LDL receptor binding site.
terminus of the 28-kDa peptide
corresponds precisely to the sequence of the apoB peptide described
above that undergoes advanced glycosylation in vitro (not
shown).
-terminal to the putative LDL receptor binding domain. Of
note, a low but detectable level of AGE modification was observed in
corresponding peptides isolated from control, native LDL, confirming
previous data showing that AGE modifications affect the LDL that
circulates in nondiabetic, non-renally impaired individuals as well
(12, 13) .
C NMR studies of
apoB have identified two classes of lysine residues that titrate with
different pK values: ``active'' lysines which have a
p K
of 8.9 and ``normal''
lysines with a p K
of 10.5
(28) .
These studies also have provided evidence for an upper limit of 21
active and 31 normal lysines within apoB that are involved in the
binding of LDL to its receptor. Nevertheless, among the proteins that
have been studied closely for the presence of Amadori (early)
glycosylation products, p K
values alone
appear to be an insufficient determinant of lysine reactivity
(29, 30) . For example, there is evidence to suggest
that the second step of the advanced glycosylation pathway, Amadori
product formation, is favored at sites of contiguous, basic residues.
The high local charge density within these motifs may suppress lysine
protonation. This would serve to increase the nucleophilicity and
reactivity of the lysine
-amino group and, at the same time,
provide adjacent protons that could participate in local acid-base
catalysis to accelerate further rearrangement reactions
(30) .
Apolipoprotein B possesses a high content of basic amino acids,
including numerous sites of contiguous, basic residue repeats. These
sites may preferentially accumulate Amadori products and also may be
favored kinetically to rapidly form intramolecular AGE cross-links,
rather than the usually occurring intermolecular AGE cross-links. The
67-amino acid domain that has been identified to undergo advanced
glycosylation within apoB contains nine potentially reactive lysine
residues and one site with three contiguous lysines
(Lys
). Whether these lysines in fact
participate in the formation of an intramolecular cross-link is not
known and would require further analysis into the chemical nature of
the apoB AGE structure. Additional microenvironment effects also may
play a role in the susceptibility of this region of apoB to advanced
glycosylation. For example, the 67-amino acid AGE reactive domain has
been mapped to a lipid-associating region of the apoB sequence
(31) , and AGE modification at this site may be favored by the
fact that anhydrous, lipophilic environments can enhance the
dehydration and rearrangement reactions leading to AGE formation
(13, 24) .
Arg substitution at codon 3500 that occurs COOH-terminal to the LDL
receptor binding domains.
C NMR analyses indicate that
this mutation perturbs the microenvironment of six lysines, changing
their average p K
values from 8.9 to 10.5
(31) . Furthermore, immunochemical studies have provided several
examples of antibodies that effectively block LDL receptor binding but
that map to epitopes spanning
1000 amino acids and that are
located in regions lying well outside the LDL receptor binding domain
(32, 33) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.