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.
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Abstract |
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Key words: apolipoprotein B100 glycosylation/atherosclerosis/ ganglioside GM3/low density lipoprotein/sialic acid
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Introduction |
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Although the concept that apoB100 glycan modifications could induce atherogenic properties on LDL was introduced more than two decades ago (Swaminathan and Aladjem, 1976; Attie et al., 1979
), this idea remains controversial (Bartlett and Stanley, 1998
; Chappey et al., 1995
; Tertov et al., 1999
; Millar, 2001
) 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., 1986
, 1989). The initial analysis of apoB100 oligosaccharide structures revealed that oligomannose and complex biantennary types are predominant (Swaminathan and Aladjem, 1976
; Taniguchi et al., 1989
). 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., 1998
; Liao et al., 1998
). Other studies in HepG2 cells have shown that inhibition of apoB100 glucose trimming by castanospermine increased proteosome-mediated apoB100 degradation (Chen et al., 1998
). 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., 1998
). 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., 1986
), however, they do not appear to play a significant role in LDL endocytosis (Shireman and Fisher, 1979
). 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., 1997) to examine the glycosylation of apoB100 derived from healthy subjects as well as hypercholesterolemic (phenotype IIa) and diabetic (noninsulin dependent) patients who are known to be at increased risk for atherosclerosis development (Ginsberg, 2000
; Illingworth, 2000
).
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Results |
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Discussion |
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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., 2000). 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, 1998
). The presence of A2G2 is not an artifact due to sample hydrazinolysis and processing as identical treatment of human
1-acid glycoprotein, which contains both mono- and disialylated complex biantennary glycans (Hermentin et al., 1992
), 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, 1985
). 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, 1997
).
Another factor contributing to the incomplete sialylation of N-glycans may be the "branch specificity" of the ß-galactoside 26 sialyltransferase (Beyer et al., 1981
). Because most of the apoB100 sialylated N-glycans were found to be present in the
26 conformation (Table II), and because it has been shown that this transferase preferentially sialylates the Gal residue on the Galß14GlcNAcß12Man
13 arm of biantennary N-glycans (Joziasse et al., 1987
), it can be predicted that the remaining arm arising from the primary Man
16 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., 1993), and because Chlamydia sp. and other microoragnisms release mucolytic enzymes (e.g., mucinases and sialidases) to perturb host defences (McGregor et al., 1994
), 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, 1981
), the release of foam cell lysosomal glycosidases (Bolton et al., 1997
) 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., 2001
).
Glycosphingolipids also contribute to the glycan content of LDL (Chatterjee and Kwiterovich, 1976; Senn et al., 1989
), and the role of both the neutral and sialylated (ganglioside) species in atherogenesis has been studied (Prokazova and Bergelson, 1994
; Bhunia et al., 1998
). In agreement with previous work (Senn et al., 1989
), we found that GM3 is the major LDL ganglioside and is present at levels of 23 mole/mole apoB100 on average. In hypercholesterolemic patients however, where hepatic apoB100 production is increased (Cummings et al., 1995
), 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, 1956
), 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., 1993
).
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., 1992; Subbaiah et al., 1999
). 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., 1998
). While the transfer of glycosphingolipids between high-densitylipoprotein (HDL) and LDL has been demonstrated (Loeb and Dawson, 1982
) 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
-tocopherol, lycopene, and
- and ß-carotene) have been found to have a similar bimodal distribution pattern throughout the LDL subpopulations (Goulinet and Chapman, 1997
), 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.
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Materials and methods |
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Plasma lipid and apolipoprotein measurements
Plasma total cholesterol (TC), triglyceride (TG) and HDL cholesterol were determined by enzymatic methods (Lepage et al., 2000). 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 = 1.0191.063 g/L) was isolated from fresh, 12-h fasted plasma (Sattler et al., 1994). Five LDL subfractions (LDL1, d = 1.0181.023; LDL2, d = 1.0231.030; LDL3, d = 1.0301.040; LDL4, d = 1.0401.051; LDL5, d = 1.0511.065 g/ml) were also isolated from selected normolipidemic and hypercholesterolemic subjects (Chapman et al., 1981
). Purity of the apoB100 was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoreses under reducing conditions using 412% 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., 1990
) in order to remove any possible contribution of apo(a) N- and O-glycans (Garner et al., 2001
) 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., 1994
). 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., 1994) 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., 1996
) 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., 1997). The indicated mixtures were incubated for 1624 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), 12 U/ml; almond meal fucosidase (AMF; EC 3.2.1.111), 3 mU/ml; bovine testes galactosidase (BTG; EC 3.2.1.23), 12 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., 1996; Rudd et al., 1997
; Garner et al., 2001
). 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, 1993). 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 1432 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., 1989), 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., 1989
). 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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