©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Major Site of Apolipoprotein B Modification by Advanced Glycosylation End Products Blocking Uptake by the Low Density Lipoprotein Receptor (*)

Richard Bucala (§) , Robert Mitchell , Kay Arnold (1), Thomas Innerarity (1), Helen Vlassara , Anthony Cerami

From the (1) Picower Institute for Medical Research, Manhasset, New York 11030 Gladstone Institute of Cardiovascular Diseases, San Francisco, California 94141

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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()(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) .

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.


MATERIALS AND METHODS

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/HO. 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/HO/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.


RESULTS

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).


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).

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) .


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.

A variety of experimental studies have established that the primary amino group of lysine (or of an -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.

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, 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.

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 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).


DISCUSSION

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-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) .

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 C NMR studies of apoB have identified two classes of lysine residues that titrate with different pK values: ``active'' lysines which have a p Kof 8.9 and ``normal'' lysines with a p Kof 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 Kvalues 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) .

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 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 Kvalues 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) .

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.


FOOTNOTES

*
This work was supported by Grant DK19655-15 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Picower Institute for Medical Research, 350 Community Dr., Manhasset, NY 11030. Tel.: 516-365-4200; Fax: 516-365-5090.

The abbreviations used are: LDL, low density lipoprotein; AGE, advanced glycosylation end product; apoB, apolipoprotein B; BHT, butylated hydroxytoluene; AGE-LDL, AGE-modified form of LDL; HPLC, high pressure liquid chromatography.


ACKNOWLEDGEMENTS

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.


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