Characterization of human apolipoprotein B100 oligosaccharides in LDL subfractions derived from normal and hyperlipidemic plasma: deficiency of {alpha}-N-acetylneuraminyllactosyl-ceramide in light and small dense LDL particles

Brett Garner1,2, David J. Harvey2, Louise Royle2, Michael Frischmann3, Fabienne Nigon4, M. John Chapman4 and Pauline M. Rudd2

2Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK; 3Institute of Medical Biology and Human Genetics, University of Innsbruck, 6020 Innsbruck, Austria; and 4INSERM Unit 321, Pavillon Benjamin Delessert, Hopital de la Pitie, 75651 Paris, Cedex 13, France

Received on April 6, 2001; revised on June 2, 2001; accepted on July 3, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The carbohydrate composition of apolipoprotein (apo) B100, particularly its degree of sialylation, may contribute to the atherogenic properties of low-density lipoprotein (LDL). We analyzed LDL apoB100 glycans derived from normolipidemic, hypercholesterolemic, and hypertriglyceridemic diabetic subjects. Using exoglycosidase carbohydrate sequencing and matrix-assisted laser desorption/ionization mass spectrometry to analyze fluorescently labeled oligosaccharides, we report evidence for several carbohydrates not previously identified on apoB100, including truncated complex biantennary N-glycans and hybrid N-glycans. The distribution and diversity of the apoB100 glycans isolated from all individuals was highly conserved. The N-glycan composition of apoB100 derived from five LDL subpopulations (LDL1, d = 1.018–1.023; LDL2, d = 1.023–1.030; LDL3, d = 1.030–1.040; LDL4, d = 1.040–1.051; LDL5, d = 1.051–1.065 g/ml) did not vary in normolipidemic or hypercholesterolemic subjects. Furthermore, we found no evidence for "desialylated" apoB100 glycans in any of the samples analyzed. Analysis of the most abundant LDL ganglioside, {alpha}-N-acetylneuraminyllactosyl-ceramide, revealed a deficiency in small dense LDL and in the most buoyant subpopulation. These data provide a novel explanation for the apparent deficiency of sialic acid in small dense LDL and indicate that the global apoB100 N-glycan composition is invariable in the patient groups studied.

Key words: apolipoprotein B100 glycosylation/atherosclerosis/ ganglioside GM3/low density lipoprotein/sialic acid


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The plasma concentration of low-density lipoprotein (LDL) is positively associated with atherosclerosis risk (Kannel et al., 1981Go). Specific biochemical modifications of apolipoprotein (apo) B100, the protein component of LDL, may also contribute to lipoprotein atherogenicity. Such modifications include partial enzymatic hydrolysis (Bhakdi et al., 1995Go), free radical mediated oxidation (Bruce et al., 1999Go), nitration, or chlorination (Heinecke, 1999Go) of the polypeptide, derivitization of lysine residues with reactive degradation products of lipid peroxidation (Steinbrecher et al., 1984Go) or glucose (Bucala et al., 1995Go), and modifications of apoB100 glycans (La Belle and Krauss, 1990Go), particularly via desialylation (Tertov et al., 1993Go). A previous study reported that apoB100 ("LDL apoprotein") derived from subjects with a predominance of atherogenic small dense LDL in their circulation was found to contain 30% less carbohydrate than normal control subjects (La Belle and Krauss, 1990Go). Watanabe heritable hyperlipidemic rabbits have also been shown to exhibit alterations in the ratio of acidic apoB100 glycans as a function of plasma cholesterol concentration (Fujioka et al., 1994Go). Other studies, in humans, have suggested that small dense LDL also comprises a desialylated LDL subfraction (Tertov et al., 1992Go; Jaakkola et al., 1993Go), the assumption being that apoB100 glycans lose terminal sialic acid residues after secretion from the liver (Tertov et al., 1998Go; Bartlett et al., 2000Go). If apoB100 desialylation occurs in vivo, this may be important as it has been shown that sialidase-treated LDL is more prone to aggregation (Tertov et al., 1989Go), exhibits increased binding to extracellular matrix components (Camejo et al., 1985Go) and can induce cellular lipid accumulation via receptor-mediated endocytosis (Filipovic et al., 1979Go; Bartlett et al., 2000Go); all factors that are thought to contribute to atherosclerotic plaque formation.

Although the concept that apoB100 glycan modifications could induce atherogenic properties on LDL was introduced more than two decades ago (Swaminathan and Aladjem, 1976Go; Attie et al., 1979Go), this idea remains controversial (Bartlett and Stanley, 1998Go; Chappey et al., 1995Go; Tertov et al., 1999Go; Millar, 2001Go) in part due to the nonspecific and, therefore, imprecise methods often used to analyze apoB100 oligosaccharide structure. The amino acid sequence of apoB100 (4563 amino acids) indicates that 16 of the 19 potential sites for N-glycosylation (containing the Asn-X-Ser/Thr sequon) are occupied (Yang et al., 1986Go, 1989). The initial analysis of apoB100 oligosaccharide structures revealed that oligomannose and complex biantennary types are predominant (Swaminathan and Aladjem, 1976Go; Taniguchi et al., 1989Go). These glycans (and their intracellular precursors) may be of functional importance. For example, treatment of HepG2 cells with tunicamycin (which inhibits protein N-glycosylation) inhibits the interaction of apoB100 with the endoplasmic reticulum chaperone calnexin, and this results in dramatically suppressed apoB100 secretion (Bonen et al., 1998Go; Liao et al., 1998Go). Other studies in HepG2 cells have shown that inhibition of apoB100 glucose trimming by castanospermine increased proteosome-mediated apoB100 degradation (Chen et al., 1998Go). Furthermore, in transgenic mice that specifically express N-acetylglucosaminyltransferase III in the liver, a bisecting GlcNAc added to the trimannosyl core of apoB glycans resulted in a dramatic inhibition of apoB secretion and concomitant hepatic accumulation of apoB and lipid (Ihara et al., 1998Go). These studies suggest that apoB100 glycans may play a role in the regulation of degradation very early in the secretory pathway. Six of the N-glycans are predicted to occur close to the LDL receptor-binding region of mature apoB100 (Yang et al., 1986Go), however, they do not appear to play a significant role in LDL endocytosis (Shireman and Fisher, 1979Go). The potential function of apoB100 glycans post hepatic secretion, therefore, remains unkown. In addition, the possible utility of variant apoB100 glycoforms as markers of atherosclerotic disease or risk remains to be assessed.

To determine whether alterations in apoB100 glycosylation could be related to atherogenic lipoprotein phenotypes, and to gain an insight into the exact structural modifications occurring with putative desialylation, the present study utilized direct and highly sensitive methodology (Rudd et al., 1997Go) to examine the glycosylation of apoB100 derived from healthy subjects as well as hypercholesterolemic (phenotype IIa) and diabetic (non–insulin dependent) patients who are known to be at increased risk for atherosclerosis development (Ginsberg, 2000Go; Illingworth, 2000Go).


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Characterization of apoB100 N-glycans
Human LDL was isolated by density gradient ultracentrifugation and apoB100 N-glycans contained therein were released by automated hydrazinolysis, fluorescently labelled with 2-aminobenzamide (2-AB) and characterized by normal phase high-performance liquid chromatography (NP-HPLC) and matrix-assisted laser desorption/ionization time of flight mass spectrometry) (MALDI-TOF MS). Figure 1 shows that 15 peaks were resolved by NP-HPLC. Exoglycosidase digestions of the glycan pool revealed that peaks 3 and 8 contained two coeluting compounds and that the trailing shoulder of peak 10 (peak 10a) was distinct from the major peak. The sequential removal of monosaccharides from the nonreducing termini of these glycans, together with a comparison of the glucose unit (GU) values of the products and precursors (Table I), enabled structural assignments to be made for all of the apoB100 N-glycans. Two major types of glycan were present: complex biantennary structures present in either a disialylated (peaks 9, 11, and 12), monosialylated (peaks 8, 10), or neutral states (peak 7), and oligomannose structures ranging in size from Man5 to Man9. The glycosidic linkage of the terminal sialic acids were mostly (91%) present in the Neu5Ac{alpha}2–6Gal conformation. Of the oligomannose structures, Man5 and Man9 were most abundant, on average accounting for 30% and 37% of the total oligomannose structures, respectively. The complex biantennary and oligomannose structures together accounted for 93% of the total glycan pool. The remaining structures (7% of the total N-glycan pool) appeared to be present as truncated forms of the biantennary glycans (peaks 2, 3, and 5, or part thereof) and hybrid structures (peaks 3, 4, and 8, or part thereof). Evidence for a small amount (0.6% of total N-glycans on average) of a disialylated triantenary complex glycan was also found as peak 14. Because of the relatively low concentrations of the latter glycans, the exoglycosidase digestions and subsequent repurification procedure did not result in detectable recovery of the predicted digestion products by HPLC in some cases (Figure 1 and Table I).



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Fig. 1. HPLC profiles of apoB100 N-linked oligosaccharides and their exoglycosidase digestion products. Human apoB100 was subjected to hydrazinolysis and the released glycans labelled with 2-AB and analyzed by NP-HPLC ("Non-digested"). The glycan pool was also treated with ABS, ABS + BTG + AMF, and JBM as indicated, to sequence the oligosaccharide chains (see Materials and methods for details). The addition of AMF, which removes outer arm fucose residues, to either ABS or ABS + BTG did not alter the HPLC retention times of the glycans. The digestion of the oligosaccharide structures to form the predicted truncated products are indicated by the solid lines where appropriate. In somes cases, all of the digestion products for the quantitatively minor peaks 2, 3, 4, and 5 were below detection levels and MS techniques were therefore used to confirm the presence of the predicted structures. Note that the addition of JBM removes only the oligomannose and hybrid structures from the total glycan profile (compare "Non-digested" to "JBM"). G.U. values were derived from a dextran ladder.

 

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Table I. apoB-100 N-linked glycan GU values
 
The structural assignment of all of the apoB100 glycans (except peak 14) was confirmed by MALDI-TOF MS (Figure 2) and the quantitatively major oligosaccharide structures were also characterized by liquid chromatography–collision-induced dissociation/mass spectrometry/mass spectromery (LC-MS/MS) using an electrospray Q-Tof mass spectrometer (Figures 3 and 4 are given as examples). The CID spectrum of the [M + 2H]2+ ion (m/z 1026.9) from the monosialylated biantennary glycan is shown in Figure 3a. B-type ions (Domon and Costello (1988)Go nomenclature) at m/z 204.1 (GlcNAc), 366.1 (Hex-GlcNAc), 528.2 (Gal-GlcNAc-Man), and 657.2 (Neu5Ac-Gal-GlcNAc) defined the composition of the antenna. Losses of the two antenna gave the Y ions at m/z 1687.5 and 1031.3. This latter ion represented the 2-AB derivative of the core pentasaccharide. Other fragments are labeled in Figure 3a. Similar B fragments were observed from the disialylated glycan (Figure 3b, c). Loss of one antenna gave the ion at m/z 1687.5 (Figure 3b), and the ion representing the derivatized pentasaccharide core following loss of the second antenna again appeared at m/z 1031.3. Fragmentation spectra for two of the five apoB100 oligomannose structures are illustrated in Figure 4.



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Fig. 2. Mass spectrum of apoB100 N-glycan pool. ApoB100 N-glycans were released by hydrazinolysis, 2-AB labeled, and analyzed by MALDI/TOF MS. The monoisotopic masses of the [M + Na]+ ion for each of the glycans is shown. Note that some of the sialylated structures are present also as Na salts. Arrows indicate O-acetylated glycans (an artifact of hydrazinolysis). The glycan structures are represented by the following symbols: open circles, mannose; closed squares, N-acetyl glucosamine; open diamonds, galactose; closed stars, N-acetyl neuraminic acid.

 


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Fig. 3. CID spectra of the [M + 2H]2+ ions from the monosialylated biantennary glycan (a), the disialylated glycan (b) and its sodium salt (c). The glycan structures are represented by the symbols given in the legend to Figure 2.

 


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Fig. 4. CID spectra of the [M + 2H]2+ ions from the oligomannose structures containing either seven (a) or eight (b) mannose residues. The glycan structures are represented by the symbols given in the legend to Figure 2.

 
Analysis of apoB100 N-glycans from different LDL preparations derived from one of the normolipemic subjects (in both the fasted and postprandial states) resulted in identical NP-HPLC profiles (data not shown). Based on the HPLC and MS data presented above, a summary of apoB100 N-glycan composition is given in Table II.


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Table II. apoB-100 N-linked glycan composition
 
Comparison of apoB100 N-glycan composition in normolipidemic and hyperlipidemic subjects
Having defined the apoB100 N-glycan composition in LDL derived from normolipidemic subjects, we then searched for potential differences in apoB100 glycosylation in hyperlipidemic patients known to be at increased risk for development of atherosclerosis. In the normolipidemic control population (N = 9), plasma cholesterol and triglyceride concentrations ranged from 3.17–6.09 mM and 0.45–1.19 mM, respectively, and we found no correlation between the minor intersample variation in apoB100 N-glycan composition observed and these lipid variables. The plasma lipid concentrations of the patients are given in Table III. Fasting glucose and glycosylated hemoglobin (HbA1c%) levels were also measured in the non–insulin-dependent diabetes mellitus (NIDDM) patients and found to be 9.6 ± 1.2 mM and 7.8 ± 0.7%, respectively (mean ± SE, N = 4). Total yields of apoB100 N-glycans after hydrazinolyis were not significantly different between patients and control subjects. A detailed analysis of all LDL apoB100 N-glycan structures (based on NP-HPLC profiles) revealed no significant differences between the patient groups or when patients were compared to the control subjects (Table II).


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Table III. Plasma lipid concentrations in control and hyperlipidemic subjects.
 
Comparison of apoB100 N-glycan composition in LDL subfractions
Previous studies, using indirect methods, have raised the possibility that plasma LDL apoB100 glycosylation may vary depending on the size of the LDL subpopulation (Tertov et al., 1992Go; Jaakkola et al., 1993Go) and in subjects with an atherogenic lipoprotein phenotype (La Belle and Krauss, 1990Go). To examine the possibility that a subfraction of LDL contains an altered apoB100 glycan profile, five major LDL subpopulations were isolated (Chapman et al., 1981Go) from both normolipidemic and hypercholesterolemic subjects. The density ranges for the LDL subpopulations as well as the relative contributions of each LDL subpopulation to total plasma LDL in both control and familial hypercholesterolemic (FH) subjects is illustrated in Figure 5. The hypercholesterolemic patients were found to have higher circulating levels of all but the lightest of the LDL subpopulations isolated. Of potential importance, the patients had two- to threefold higher concentrations of small dense LDL (LDL4 and LDL5) compared to the controls (Figure 5). When apoB100 N-glycans were analyzed separately in all five LDL subpopulations, no significant differences were found either between the LDL subpopulations or comparing the hypercholesterolemic patients to the control subjects (Table IV). Hydrazinolysis of apoB100 also generated a small amount (1 mole per mole apoB100) of oligosaccharides ranging in size from two to four monosaccharide residues, consistent with the presence of O-linked glycans (data not shown). We did not detect any consistent differences in the contents of these putative O-glycans when patients were compared to normolipemic subjects (Garner et al., unpublished data).



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Fig. 5. LDL subpopulation profiles in control and FH patients. LDL was isolated from fasted plasma into five density subpopulations by ultracentrifugation. The relative contribution of each subpopulation to total plasma LDL is indicated by the LDL protein concentration of each fraction. The white bars represent normolipemic control subjects and the black represent heterozygous familial hypercholesterolemic patients. Data are means, error bars represent SE (N = 3 for each group). *P < 0.05; **P < 0.01.

 

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Table IV. apoB100 N-linked glycan composition in LDL subpopulations
 
Distribution of GM3 in plasma and LDL subfractions
Our data showing that the degree of sialylation of apoB100 N-glycans remains constant when comparing control subjects to hypercholesterolemic and NIDDM patients and throughout the range of LDL subpopulations appears to contradict earlier reports (Tertov et al., 1992Go; Jaakkola et al., 1993Go; Sobenin et al., 1993Go, 1996). One important difference between our study and earlier work is that we have used direct methods to analyze glycan structure, whereas previous work has often relied on more convenient but less specific chemical methods (Sobenin et al., 1998Go). Because LDL is the major plasma transporter for the sialylated glycosphingolipids (gangliosides) we investigated the possibility that the sialic acid content of LDL could vary due to its ganglioside content. In agreement with earlier work (Senn et al., 1989Go), we found that the major LDL ganglioside was Neu5Ac{alpha}2–3Galß1–4Glcß1-ceramide, GM3 (Figure 6). The identity of GM3 was confirmed by exoglycosidase sequencing of the released glycan and by its coelution from the NP-HPLC column with the glycan moiety of a pure GM3 standard (data not shown). Plasma GM3 concentration ranged from approximately 4 to 8 µM, in general agreement with previously reported values (Kundu et al., 1985Go), and was correlated with plasma cholesterol in the subset of our study population examined (Figure 7). Across the range of values observed, a twofold increase in plasma cholesterol was correlated with a twofold increase in GM3 levels (note that the absolute concentrations were 1000-fold lower for GM3). We then analyzed GM3 levels in LDL derived from the control and FH patients in more detail. Despite the higher GM3 levels found in the hypercholesterolemic plasma, when plasma GM3 concentration was expressed as the GM3:apoB100 ratio (which also approximates the GM3 per LDL particle ratio), the ratio was found to be significantly lower in the hypercholesterolemic subjects, that is, the plasma apoB100 concentrations were 0.697 ± 0.138 and 1.570 ± 0.070 mg/ml in the control and FH subjects, respectively, and the resulting GM3:apoB100 ratios were 7.84 ± 0.78 and 4.44 ± 0.47 nmol/mg, respectively (all values mean ± SE, N = 3). This implied that the hypercholesterolemic LDL or (a specific subpopulation) was sialic acid deficient. This was addressed directly by analysis of the same LDL subpopulations used for the apoB100 N-glycan analysis above.



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Fig. 6. HPLC profile of LDL ganglioside composition. LDL gangliosides were isolated by solvent extraction and the glycan portion released by ceramide glycanase treatment. The released glycan pool was then 2-AB labeled and analyzed by NP-HPLC. The major ganglioside present was GM3 and its identity was confirmed by exoglycosidase digestions with ABS, BTG, and SPH and by spiking samples with 2-AB-labeled glycans derived from a pure commercially available GM3 standard (not shown). The glycan structures are represented by the symbols described in the legend to Figure 2 (open squares, glucose).

 


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Fig. 7. Correlation of plasma cholesterol and GM3 concentrations. Plasma cholesterol concentration was measured enzymatically and GM3 concentration measured after removal of the glycan moiety, using ceramide glycanase, then labeling with 2-AB and quantitation by NP-HPLC. The individual points on the graph represent each of the subjects studied. FH, familial hypercholesterolemic (heterozygous, type IIa); NIDDM, non–insulin-dependent diabetes mellitus; Con, normolipidemic control.

 
Figure 8 shows that there were striking differences in the distribution of GM3 throughout the LDL density subpopulations. Interestingly, the composition of the particles was very similar when the controls were compared to the FH subjects; with both the dense and light subpopulations containing less GM3 per particle on average. However, because the hypercholesterolemic patients’ plasma contained a two- to threefold higher concentration of the dense LDL subpopulations (Figure 5), their plasma also contains a higher concentration of this sialic acid deficient LDL. Importantly, this sialic acid deficit is not due to GM3 desilaylation, as the amount of product, lactosyl ceramide, was not increased in the dense LDL subpopulations compared to the other populations (data not shown).



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Fig. 8. Distribution of GM3 in LDL subpopulations. The LDL subpopulations isolated as illustrated in Figure 3 were analyzed for GM3 by HPLC as described in the legend to Figure 6. The GM3 concentration was expressed per mole of apoB100 for each LDL fraction. The white bars represent normolipidemic control subjects and the black represent heterozygous familial hypercholesterolemic patients. Data are means, error bars represent range (N = 2 for each group).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The carbohydrate moiety of many glycoproteins plays an important role in their folding, stability and biological function (Rudd and Dwek, 1997Go). In some cases, altered physiological states can induce alterations in protein glycosylation. For example, in rheumatoid arthritis patients, an altered IgG glycosylation pattern can serve as a marker for disease (Parekh et al., 1985Go). Similarly, glycosylation of {alpha}1-acid glycoprotein is altered in rheumatoid arthritis and other inflammatory disorders (Ryden et al., 1997Go). Previously it has been reported that the apoB100 carbohydrate content can vary from 4% to 10% by mass (see La Belle and Krauss, 1990Go and references cited therein) and that a small dense subfraction of LDL in normal and hyperlipidemic plasma is desialylated (Jaakkola et al., 1993Go). Here we sought to elucidate the potential modifications of apoB100 glycans to understand better how the oligosaccharide structure might be related to the atherogenic properties of LDL or its subpopulations. The initial characterization of apoB100 glycans (Swaminathan and Aladjem, 1976Go; Taniguchi et al., 1989Go) has, therefore, been extended here where the use of more sensitive techniques revealed the full compliment of neutral, mono-, and disialylated biantennary complex glycans, along with the linkage analysis of their sialylated antennae, which has not been previously determined. We also detected several classes of oligomannose and three hybrid structures (Table II). Given that human apoB100 has a relative molecular mass of 512,937 and that 16 Asn residues are occupied by N-glycans (Yang et al., 1986Go, 1989), the data in Table II indicate that apoB100 contains 6.3% carbohydrate by mass.

The novel discovery that all of the samples analyzed contained the neutral complex biantennary glycan (abbreviated here as A2G2), is potentially significant because it is a likely ligand for a macrophage asialoglycoprotein receptor that is involved in foam cell formation (Bartlett et al., 2000Go). This finding is also consistent with data showing that all LDL particles can bind to the Gal-specific lectin, Ricinus communis agglutinin I (Bartlett and Stanley, 1998Go). The presence of A2G2 is not an artifact due to sample hydrazinolysis and processing as identical treatment of human {alpha}1-acid glycoprotein, which contains both mono- and disialylated complex biantennary glycans (Hermentin et al., 1992Go), did not result in A2G2 formation (data not shown). In addition, when apoB100 N-glycans were removed by peptide N-glycosidase digestion, A2G2 was still detected (data not shown). Complex biantennary N-glycans can be secreted in either a neutral, monosialylated, or disialylated state (Kornfeld and Kornfeld, 1985Go). This variable sialylation is at least partially determined by the local 3D structure of the nascent glycoprotein and the access of glycans attached to specifc domains of sialyltransferases in the Golgi membrane (Rudd and Dwek, 1997Go).

Another factor contributing to the incomplete sialylation of N-glycans may be the "branch specificity" of the ß-galactoside {alpha}2–6 sialyltransferase (Beyer et al., 1981Go). Because most of the apoB100 sialylated N-glycans were found to be present in the {alpha}2–6 conformation (Table II), and because it has been shown that this transferase preferentially sialylates the Gal residue on the Galß1–4GlcNAcß1–2Man{alpha}1–3 arm of biantennary N-glycans (Joziasse et al., 1987Go), it can be predicted that the remaining arm arising from the primary Man{alpha}1–6 linkage is more likely to remain in a neutral state. We therefore suggest, based on the published experimental evidence and the data included herein, that the term "desialylated LDL" as applied to differences in human apoB100 N-glycans derived from normolipidemic, diabetic, and hypercholesterolemic LDL and its subpopulations in plasma may be a misnomer as the occurrence of N-linked glycans with terminal Gal residues could also reflect normal apoB100 glycan processing in the Golgi.

It is possible that desialylation of apoB100 glycans does occur at specific sites under certain pathological conditions. For example, because a significant proportion of atherosclerotic lesions contain Chlamydia pneumoniae (Kuo et al., 1993Go), and because Chlamydia sp. and other microoragnisms release mucolytic enzymes (e.g., mucinases and sialidases) to perturb host defences (McGregor et al., 1994Go), desialylation of apoB100 glycans may occur in the artery wall where such microbes exist. In addition, in advanced atherosclerotic lesions, where a large number of macrophage foam cells are present (Gerrity, 1981Go), the release of foam cell lysosomal glycosidases (Bolton et al., 1997Go) might alter apoB100 glycan structure. It is also possible that sialic acid is present in structures that we have not detected. Of potential significance, preliminary data suggests that a mammalian trans-sialidase may use polysialic acid structures as a substrate for lipoprotein sialic acid transfer (Tertov et al., 2001Go).

Glycosphingolipids also contribute to the glycan content of LDL (Chatterjee and Kwiterovich, 1976Go; Senn et al., 1989Go), and the role of both the neutral and sialylated (ganglioside) species in atherogenesis has been studied (Prokazova and Bergelson, 1994Go; Bhunia et al., 1998Go). In agreement with previous work (Senn et al., 1989Go), we found that GM3 is the major LDL ganglioside and is present at levels of 2–3 mole/mole apoB100 on average. In hypercholesterolemic patients however, where hepatic apoB100 production is increased (Cummings et al., 1995Go), the average content of GM3/LDL particle was found to be reduced by 43% in the present study. Furthermore, when LDL subpopulation analysis was performed, we found a bimodal distribution of GM3 in the LDL subpopulations; with the light LDL1 and dense LDL5 subfractions containing approximately one-third of the amount of GM3 found in the major LDL3 subpopulation (Figure 8). Because ganglioside sialic acid residues are removed using the conditions of mild acid hydrolysis typically employed to measure LDL sialic acid levels (Svennerholm, 1956Go), we suggest that differences in GM3 distribution in the different density subpopulations confound the data suggesting that dense LDL subpopulations are desialylated. Our data showing that GM3 levels are lower in the light and dense LDL subfractions in both control and hyperlipidemic subjects bear a striking resemblance to results published in a previous study that implied that the dense subpopulations were desialylated (Jaakkola et al., 1993Go).

The reasons for the lower content of GM3 in small dense LDL, when expressed per particle (or per mole apoB100 or mg apoB100), are readily explained by the fact that glycosphingolipids are anchored into the surface of LDL by their lipophylic ceramide moiety and, because small dense LDL have a smaller surface area compared with the major LDL subpopulations, the relative GM3 content is predicted to be lower; as is the case for LDL phospholipids including sphingomyelin (Liu et al., 1992Go; Subbaiah et al., 1999Go). The relatively low level of GM3 in the larger buoyant LDL1 subpopulation was not predicted. This could be a reflection of preferential transfer of gangliosides between lipoprotein species. It is known that cholesteryl ester transfer rates to different triglyceride-rich lipoprotein species vary markedly due to the activity of cholesteryl ester transfer protein (Lassel et al., 1998Go). While the transfer of glycosphingolipids between high-densitylipoprotein (HDL) and LDL has been demonstrated (Loeb and Dawson, 1982Go) no data are available concerning possible preferential transport between lipoprotein subpopulations and whether or not this is facilitated by transport proteins in vivo. Interestingly, other LDL constituents (including {gamma}-tocopherol, lycopene, and {alpha}- and ß-carotene) have been found to have a similar bimodal distribution pattern throughout the LDL subpopulations (Goulinet and Chapman, 1997Go), however no mechanistic explanation for this exists.

In conclusion, a detailed analysis of human apoB100 N-glycans indicates that average glycosylation levels and distribution of structures is highly conserved in normal subjects and in selected individuals known to be at high risk for atherosclerosis development. Glycans containing terminal Gal residues appear to be a normal component of LDL N-glycans and, based on current experimental evidence, there is no reason to believe that they are the result of desialylation in the plasma compartment. The bimodal distribution of GM3 throughout the LDL density subpopulations, particularly its relative deficiency in small dense LDL, provides a plausible explanation for at least part of the apparent sialic acid deficiency that has previously been associated with small dense LDL.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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 Chemicals (Loughborough, UK). Phosphate-buffered saline was prepared using Oxoid (Basingstoke, UK) tablets. All reagents for hydrazinolysis were from Oxford GlycoSciences (Abingdon, UK). All exoglycosidases were from Glyko (Novato, CA), except ceramide glycanase, which was from Calbiochem (La Jolla, CA). All other reagents were of the highest quality available and purchased from Sigma (St. Louis, MO).

Plasma lipid and apolipoprotein measurements
Plasma total cholesterol (TC), triglyceride (TG) and HDL cholesterol were determined by enzymatic methods (Lepage et al., 2000Go). LDL cholesterol was calculated according to the Friedwald formula (LDL cholesterol = TC – HDL cholesterol – [TG/5], mM). ApoB100 and lipoprotein(a) were measured by laser immunonephelometry on a Behrin BNA nephelometer analyzer (Rueil Malmaison, France).

Isolation of LDL and apoB100
Total LDL (d = 1.019–1.063 g/L) was isolated from fresh, 12-h fasted plasma (Sattler et al., 1994Go). Five LDL subfractions (LDL1, d = 1.018–1.023; LDL2, d = 1.023–1.030; LDL3, d = 1.030–1.040; LDL4, d = 1.040–1.051; LDL5, d = 1.051–1.065 g/ml) were also isolated from selected normolipidemic and hypercholesterolemic subjects (Chapman et al., 1981Go). Purity of the apoB100 was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoreses under reducing conditions using 4–12% Tris-glycine gradient gels with Coomassie or silver staining (Novex, San Diego, CA). In specific experiments, apo(a)-free apoB100 was specially prepared (Menzel et al., 1990Go) in order to remove any possible contribution of apo(a) N- and O-glycans (Garner et al., 2001Go) to the apoB100 sample. In brief, 480 ml of plasma was adjusted to d = 1.063 g/ml with KBr and 48 µg merthiolate, 96 µg NaN3, 48 µl butylated hydroxy toluene, 480 µl 0.5 M Na2EDTA, and 225 µl phenylmethylsulfonyl fluoride were added. The LDL plus very-low-density lipoprotein fraction was then isolated by sequential ultracentrifugation first in a Beckman 50.2 rotor (49,000 r.p.m., 48 h, at 10°C). The very-low-density lipoprotein fraction was then removed after adjusting to d = 1.001 g/ml and spinning in the 50.2 rotor (40,2000 r.p.m., 24 h, at 10°C), and finally the LDL fraction was collected and adjusted to d = 1.021 g/ml with KBr and subjected to density gradient ultracentrifugation in a Beckman SW41 rotor (40,000 r.p.m., 24 h, at 10°C). Fractions of LDL (400 µl per fraction) were removed and assayed for apoB100 and apo(a) by an enzyme-linked immunosorbent assay described previously (Kronenberg et al., 1994Go). The apo(a)-free LDL fraction contained apoB100 at a concentration of 0.5 mg/ml and did not contain detectable apo(a) (limit of sensitivity 0.10 µg/ml).

Release and fluorescent labeling of apoB100 N-linked oligosaccharides
Isolated LDL (100 µg protein) or apoB100 delipidated with hexane/methanol (Sattler et al., 1994Go) was subjected to automated hydrazinolysis using a GlycoPrep 1000 instrument (Oxford GlycoSciences Ltd.). For the N-glycan release, hydrazinolysis was performed at 100°C for 5 h to achieve maximal recovery. The glycan profiles of native and delipidated apoB100 were identical, therefore, delipidation was not routinely performed. The recovered glycan solutions were evaporated to dryness using a vacuum centrifuge and their reducing termini fluorescently labeled by reductive amination (Townsend et al., 1996Go) with 2-AB using a LudgerTag kit (Ludger, Oxford, UK).

Simultaneous exoglycosidase sequencing of the released glycan pool
The 2-AB labeled glycan pools (not the individual peaks) were evaporated to dryness and arrays of standardized enzyme solutions were added to aliquots of each sample (Rudd et al., 1997Go). The indicated mixtures were incubated for 16–24 h at 37°C in 50 mM sodium acetate buffer (pH 5.5) or 100 mM sodium acetate buffer (pH 5.0) containing 2 mM zinc acetate where mannosidase was used. Conditions for the individual enzymes in the arrays were as follows: Atherobacter ureafaciens sialidase (ABS; EC 3.2.1.18), 1–2 U/ml; almond meal fucosidase (AMF; EC 3.2.1.111), 3 mU/ml; bovine testes galactosidase (BTG; EC 3.2.1.23), 1–2 U/ml; Streptococcus pneumoniae hexosaminidase (SPH; EC 3.2.1.30), 2 U/ml; Newcastle disease virus sialidase (NDVS; Hitchner B1 strain; EC 3.2.1.18), 0.2 U/ml; jackbean mannosidase (JBM; EC 3.2.1.24) 5 U/ml. Samples were purified from the exoglycosidases before HPLC analysis by passing them through a microcentrifuge tube inset with a protein binding filter (Microspin 45 µ CN, Pro-Mem, R.B. Radley and Co Ltd., Shire Hill, UK). Exoglycosidase digestions were performed on apoB100 N-glycans derived from two normolipidemic subjects.

Analysis of N-Glycans by NP-HPLC
HPLC separations were performed on a 0.46 x 25 cm Glycosep-N chromatography column (Oxford GlycoSciences) using the low-salt conditions and structures were assigned by comparision of their elution positions (converted to GU values) with known GU values, for previously sequenced glycans, both before and after exoglycosidase digestions as described previously (Guile et al., 1996Go; Rudd et al., 1997Go; Garner et al., 2001Go). NP-HPLC analysis of apoB100 N-glycans was conducted using glycans derived from nine normolipemic, three heterozygous type IIa hypercholesterolemic, one homozygous type IIa hypercholesterolemic, and four NIDDM subjects.

Analysis of N-glycans by MALDI-TOF and LC-MS/MS
Positive ion MALDI-TOF mass spectra were recorded with a Micromass TofSpec 2E reflectron-TOF mass spectrometer (Micromass 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 recrystallised from ethanol (Harvey, 1993Go). Samples were purified from residual peptides and salts prior to MALDI-TOF MS by passing them through mixed bed resins of Chelex100 (Na+)/Dowex AG50X 12 (H+)/Dowex Ag3X 4A (OH-)/QAE Sephadex A-25. MALDI-TOF MS characterization of apoB100 N-glycans was performed twice using apoB100 derived from two normolipemic individuals.

Fragmentation spectra of the major N-linked glycans were acquired with an electrospray Q-Tof mass spectrometer (Micromass) fitted with a nanoflow probe and a Z-spray ion source and interfaced to a waters Cap-LC HPLC system. The glycans were separated on an NP-HPLC column using the above conditions. The mass spectrometer was set up as follows: the electrospray ion source, 100°C; desolvation gas, 120°C; capillary voltage, 3.0 kV; cone voltage, 30 V; collision gas, argon at 20 psi; collision energy, appropriate to the mass of the glycan but in the region of 14–32 V; spectra accumulated for 2.4 s. The instrument was used in auto-CID mode with three channels being monitored. After the intensity of each detected ion had dropped below a preset level, that mass was excluded from the channel for a period of 30 s. MS/MS spectra were recovered by averaging all scans for a particular parent ion from each channel.

Measurement of plasma and LDL GM3 concentrations
Forty microliters of human plasma or LDL (1 mg protein/ml) was extracted in 4 ml chloroform/methanol (2:1 v/v) by 15-s vigorous shaking in a glass tube followed by 15 min sonication in a water bath at 22°C. The samples were then placed on ice for approximately 2 min and subsequently centrifuged at 4000 r.p.m. for 10 min at 4°C in a Beckman Allegra 6R centrifuge. The supernatant (3.6 ml) was removed and evaporated to dryness under vacuum (Howe, GyroVap). The crude lipid fraction was redissolved in 200 µl chloroform and passed over a silicic acid column (prepared by adding 1 ml of silicic acid/chloroform suspension [10% w/v] to a plastic cartridge containing a frit at the outlet). The lipid fractions were eluted from the column in the following order: 5 ml chloroform was used to elute neutral lipids (predominantly cholesteryl esters and triglycerides); 1.8 ml of methanol/acetone (1:9 v/v) was then used to elute neutral glycolipids (predominantly lactosyl ceramide and glucosyl ceramide); and the ganglioside fraction (predominantly GM3) was eluted with 1.8 ml methanol. The ganglioside fraction was evaporated to dryness under vacuum and redissolved in 50 µl of 50 mM sodium actetate buffer (pH 5) containing 1 mg/ml sodium cholate and 0.1 U ceramide glycanase. The mixtures were then incubated in a sterile atmosphere at 37°C for 18 h to release the carbohydrate moiety from ceramide (Zhou et al., 1989Go), passed over an Oasis HLB cartridge (Waters), fluorescently labeled with 2-AB, and analyzed by NP-HPLC as described. Under these conditions GM3 is completely hydrolyzed as assessed by thin-layer chromatography and scanning densitometry (Zhou et al., 1989Go). The quantitation of GM3, by comparison with a commercial standard (Sigma), was accurate over a linear range from 1 pmol to at least 10 µmol. Recovery of exogenously added GM3 was assessed in three experiments and found to be 75 ± 10% (mean ± SE, D. R.Wing and B. Garner, unpublished data).

Statistical analysis
Statistical significance was determined using the two-tailed t-test for unpaired data. A P value < 0.05 was considered significant.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by a Wellcome Trust fellowship and grant (058833) awarded to B.G. The authors wish to thank Dr. Fredrick Karp for supplying NIDDM plasma and Prof. Raymond A. Dwek for helpful comments on the manuscript. We also thank the Biotechnology and Biological Sciences Research Council and the Higher Education Funding Council for England for funding the mass spectrometers.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
2-AB, 2-aminobenzamide; ABS, Atherobacter ureafaciens sialidase; A2G0, nonsialylated complex biantennary glycan containing no galactose; A2G2, nonsialylated complex biantennary glycan containing two galactose residues; A2G2S1, monosialylated complex biantennary glycan containing two galactose residues; A2G2S2, disialylated complex biantennary glycan containing two galactose residues; AMF, almond meal fucosidase; apoB100, apolipoprotein B100; BTG, bovine testes galactosidase; FH, familial hypercholesterolemia; CID, collision-induced dissociation; GM3, ganglioside {alpha}-N-acetylneuraminyllactosyl-ceramide; GU, glucose unit; HDL, high-density lipoprotein; JBM, jackbean mannosidase; LDL, low-density lipoprotein; LC-MS/MS, liquid chromatorgraphy–CID mass spectrometry/mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; NIDDM; non–insulin-dependent diabetes mellitus; NP-HPLC, normal phase high-performance liquid chromatography; SPH, Streptococcus pneumoniae hexosaminidase; TC, total cholesterol; TG, triglyceride.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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