2 UMR 8576 Du Cnrs, Laboratoire De Glycobiologie Structurale Et Fonctionnelle, Et Ifr 118 Ustl, 59655, Villeneuve D'ascq, France
3 Clinique De La Charité, Chru De Lille, 59045, Lille, France
4 Laboratoire De Biochimie Et De Biologie Moléculaire, Hôpital Calmette Bld Du Professeur Jules Leclerc, Lille 59037 Cedex, France
Received on July 9, 2002; revised on September 23, 2002; accepted on September 23, 2002
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Abstract |
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Key words: alcohol / carbohydrate-deficient transferrin / hyposialylation
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Introduction |
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The presence of a hyposialylated form of transferrin in the plasma of chronic alcohol consumers was described 25 years ago by Stibler and termed carbohydrate-deficient transferrin (CDT). Due to the sensitivity and the specificity of CDT, it is considered the best marker available of chronic alcohol consumption. More than 150 studies have been performed on CDT as a marker of heavy drinking. However the use of this marker is still controversial (Fagerberg et al., 1994; Schmitt et al., 1998
; Allen and Sillanaukee, 1999
). The mechanisms of transferrin hyposialylation in chronic alcohol intake are not well understood. Ethanol has been shown to modify both the biosynthesis and the catabolism of glycoproteins (Stibler and Borg, 1991
; Ghosh et al., 1993
; Xin et al., 1995
; Cottalasso et al., 1996
; Clemens et al., 1996
; Tworek et al., 1996
; Lakshman et al., 1999
). Therefore, the presence of CDT in the serum of chronic alcohol drinkers should correspond to the sum of all the hyposialylation mechanisms, and the oligosaccharide moiety of transferrin should reflect the involvement of each of them. Conversely, the only modification of carbohydrate deficient transferrin described in the literature is the loss of one or both glycan chains (Landberg et al., 1995
; Peter et al., 1998
; Inoue et al., 1999
).
The goal of the present work was to identify the structure of the oligosaccharides present on each isoform of transferrin. The knowledge of the involvement of the various hyposialylation mechanisms might be of importance in the understanding of the presence or the absence of the hyposialylated transferrin during chronic alcohol intake as well as in determining if this marker can be used to distinguish signs of alcohol consumption and of alcohol related damage.
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Results |
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Transferrin isoforms were separated by ion-exchange chromatography. Three fractions (F1, F2, and F3) were isolated in the serum of the healthy subject (Figure 1A). The protein content of each fraction was evaluated with BCA assay and analyzed by IEF: fraction F1 contains mainly trisialylated isoform with a minor tetrasialylated isoform contamination; fraction F2 contains tetrasialylated isoform and a minor amount of pentasialylated transferrin; and fraction F3 includes penta- and hexasialylated isoforms.
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The released N-glycans from each transferrin isoform were first analyzed by fluorophore-assisted carbohydrate electrophoresis (Jackson, 1994). The electrophoretic profiles are shown in Figure 1B. The relative mobility of the 8-aminonaphthalene-1,3,6,-trisulfonate (ANTS) oligosaccharide fluorescent derivatives, expressed as the relative migration index (RMI) (Stack and Sullivan, 1992
), was evaluated using a ladder of maltooligosaccharides. The major and most anodic electrophoretic band of fraction F2 with a RMI of 6.7 was eluted from the gel and studied by matrix-assisted laser desorption and ionization mass spectrometry (MALDI MS). The mass spectrum only showed ions at m/z 2588 corresponding to an ANTS-derived oligosaccharide. The deduced monosaccharide composition corresponded to Hex5HexNAc4NeuAc2 (Klein et al., 1998
). Interpretation of the electrophoretic profiles was completed with the RMI of the different ANTS-derived oligosaccharides (Stack and Sullivan, 1992
; Hu, 1995
).
The electrophoretic profile of ANTS-labeled oligosaccharides of fraction F3 contains a major band with a RMI of 6.7 and a broad band with a RMI between 8 and 9 that is absent of fractions F1 and F2 and corresponds to triantennary trisialylated oligosaccharides. Numerous minor bands are also observed with RMI ranging from 7 to 8. The oligosaccharide electrophoretic pattern of fraction F3, corresponding to penta- and hexasialotransferrins, is in agreement with the results of other groups and confirms that these isoforms are principally composed of the association either of a biantennary and a triantennary or of two triantennary fully sialylated oligosaccharides (de Jong et al., 1990; Landberg et al., 1995
). Fraction F2 is constituted principally of biantennary bisialylated oligosaccharides; fraction F1 mainly contains biantennary bisialylated oligosaccharides and two other bands having a slower mobility (RMI between 7.0 and 7.5) that probably correspond to monosialylated biantennary oligosaccharides or to monofucosylated biantennary oligosaccharides (Stack and Sullivan, 1992
). Nevertheless, all the observed bands could not be assigned to a precise oligosaccharide structure, and a mass spectrometric approach was performed to complete the analysis.
To avoid fragmentation by loss of terminal sialic acid, we used a method involving methylesterification of the carboxyl group, which allows the simultaneous analysis of neutral and sialylated oligosaccharides in the positive-ion detection mode (Powell and Harvey, 1996). As shown in Figure 2, the major ions at m/z 2274 present in all the studied fractions correspond to an oligosaccharide with a chemical composition of NeuAcMe2Hex5HexNAc4. The monosaccharide composition of each observed ion in the different fractions is summarized in Table I. The relative intensity of ions elucidates the combination of the two N-glycans present in each isoform. Fraction F1 contains mainly trisialotransferrin and, as shown by the two major ions seen in the mass spectrum, is the result of the association of a disialylated (m/z 2274) and a monosialylated (m/z 1969) biantennary oligosaccharide (Figure 2A). Minor ions at m/z 2420 correspond to a disialylated and fucosylated biantennary oligosaccharide. Fraction F2 contains mainly ions at m/z 2274, indicating that the association of two disialylated biantennary glycans constitutes the major part of tetrasialotransferrin. The distribution of negative charges due to the presence of sialic acids on the two oligosaccharides is 2/2. The minor ions at m/z 1969, corresponding to a monosialylated biantennary oligosaccharide, might have two different origins. It originates either from the presence of trisialotransferrin contaminating the fraction F2 (due to the limits of the resolution of the high-performance liquid chromatography column resulting in overlapping fractions) or from some tetrasialotransferrin having a 3/1 charge repartition on the two glycans. However the ions at m/z 2944 are weakly detected, suggesting a purification procedure origin.
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Fraction F3 (Figure 2C) differs from the other by the presence of intense ions at m/z 2944 and 3090 corresponding to a triantennary trisialylated oligosaccharide, fucosylated or not. The repartition of the charge due to sialic acids on transferrin in fraction F3 is 2/3. Presence of ions at m/z 1969 might have two origins, either the association of a monosialylated N-glycan with a tetraantennary tetrasialylated oligosaccharide as indicated by the minor ions at m/z 3614 or the methylesterified sialic acids undergo a residual desialylation on the target during ionization in spite of the stabilizing effect of the chemical modification.
Purification of transferrin isoforms from patients with severe alcohol abuse
Human transferrin was isolated from the serum using the procedure described previously. Four fractions (F0, F1, F2, and F3) were obtained by ion-exchange chromatography (Figure 3A). IEF of the different fractions indicated that fraction F0 contains exclusively disialylated transferrin and that the electrophoretic profiles of the other fractions were similar to the corresponding fractions isolated from the control subjects.
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The MS analysis indicates that the CDT fraction F0 principally contains the ions at m/z 2274 (Figure 4A), suggesting that this isoform is mainly originated from the absence of a complete oligosaccharidic chain. Nevertheless, a minor peak at m/z 1664 indicated the possible combination of two biantennary oligosaccharides with a charge repartition of 2/0. Finally, the ions at m/z 1969 indicate that a minor disialotransferrin glycoform is constituted with two monosialylated biantennary oligosaccharides with a charge repartition 1/1.
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In the digestion with both galactosidase and neuraminidase (Figure 5A), the two ions are found at m/z 1543 for the triantennary structure and at 1705 for the biantennary structure with a ratio of 10/1. In the digestion of methylesterified oligosaccharides with galactosidase (Figure 5B), ions at m/z 2010 (generated by the triantennary structure) and 2172 (generated by the biantennary structure) are detected in a similar ratio. From these experiments the original ions at m/z 2334 probably correspond to a mixture of a major compound, a triantennary monosialylated and a minor one, a biantennary monosialylated with an additionnal N-acetyllactosamine unit. These ions are also present in the tetrasialotransferrin fraction (F2).
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Discussion |
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An interesting observation is the absence of a triantennary trisialylated oligosaccharide in the trisialotransferrin fraction (F1), suggesting the absence of a molecule constituted of a single oligosaccharide chain in this fraction. The lack of this glycoform of transferrin can be either an artifact of the analytical process, the chromatographic behavior on the MonoQ column of a trisialotransferrin constituted with a single oligosaccharide chain might be different from the other trisialylated isoforms, or the fact that during the processing of the oligosaccharide the N-acetyl glucosamine V transferase cannot function on a transferrin possessing a single chain.
The loss of an entire oligosaccharide chain is not the only modification of glycosylation observed during chronic alcohol intake. Alteration of terminal sialylation is demonstrated by the presence of (1) monosialylated N-glycan in the disialotransferrin fraction (F0); this structure also indicates that a minor fraction of CDT is a glycoprotein possessing two monosialylated biantennary N-glycans and (2) monosialylated triantennary oligosaccharide in the tri- and in the tetrasialotransferrin (F1 and F2).
In the mass spectra, the intensity of ions gives only a rough approximation of the relative quantities of each glycan. Nevertheless, the ratio of ions corresponding to monosialylated biantennary N-glycans (m/z 1969) present in tri- (fraction F1) and tetrasialylated transferrins (F2) appears similar when compared between controls and patients with severe alcohol abuse (compare mass spectra of Figure 2A with Figure 4B and Figure 2B with Figure 4C). Furthermore, there is not a major increase in the sera of the trisialotransferrin fraction (F1) relative to the tetra- and pentasialylated forms (F2 and F3).
In a recent observation, Dibbelt (2000) noticed that an increase of disialotransferrin was not accompanied by an increase of trisialotransferrin. This observation led the author to the conclusion that it is not the terminal sialylation of transferrin that is impaired in chronic alcohol intake (Dibbelt, 2000
). Nevertheless, chronic alcohol intake has been shown to be responsible for alterations that should lead to an abnormal terminal sialylation. A decreased level of sialyltransferase activity has been observed during chronic alcohol consumption with a destabilisation of sialyltransferase mRNAs (Cottalasso et al., 1996
; Lakshman et al., 1999
; Rao and Lakshman, 1999
). An increased activity of a hepatocyte membrane associated sialidase has also been described (Xin et al., 1995
). As a result of these two mechanisms, an increase of transferrin oligosaccharides with terminal galactose residues should be observed. Furthermore, the catabolism of transferrin might influence the distribution of the different isoforms. Senescent glycoproteins, which have lost terminal sialic acids, are cleared from the serum by the hepatocytes; one of the pathways is mediated by the asialoglycoprotein receptor (Ashwell and Harford, 1982
; Tozawa et al., 2001
). Chronic alcohol intake impairs multiple aspects of the receptor mediated endocytosis (McVicker and Casey, 1999
); the cellular redistribution and the altered biosynthesis of the asialoglycoprotein receptor (AGPR) have been demonstrated (Tworek et al., 1996
). Modifications of the biosynthesis and of the catabolism of transferrin through chronic alcohol intake are complex and result from the sum of multiple and sometimes opposite effects. Monosialotransferrin is not detected by IEF or ion-exchange chromatography, disialotransferrin (F0) is mainly constituted of a transferrin with a single oligosaccharide fully sialylated, and the trisialotransferrin fraction (F1) is not increased during chronic alcohol intake. All these phenomena might be the result of the elimination rate of the hyposialylated forms with terminal galactose residues by the hepatocytes, despite the partial alteration of the receptor-mediated endocytosis. Each glycovariant of transferrin has probably a different affinity toward the AGPR, and this affinity is dependent on the number of terminal galactose and sialic acid residues. A precise quantification of the terminal galactose residues in the most acidic transferrin might indicate the intensity of the phenomenon. Identification and quantification of the various alterations of the glycosylation are important to distinguish the markers of chronic alcohol consumption and of alcohol-related damages.
Furthermore, an interesting observation is that some patients with CDG type Ia signs and symptoms have a normal distribution of transferrin isoforms with decreased enzyme activities (Dupre et al., 2001). The relatively poor sensitivity of detection of chronic alcohol consumption by the measurement of CDT can probably be explained by genetic factors related to carbohydrate (Freeze, 2001
) and ethanol metabolism (Yoshida, 1994
).
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Materials and methods |
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Patient samples
Serums from patients with chronic alcohol abuse were collected in an alcohol-dependency clinic (Clinique de la Charité, CHRU de Lille). Control serum was obtained from volunteers.
IEF
Serums were saturated and diluted (1:20) with ferric citrate (40 µM) in the presence of sodium bicarbonate (50 mM) and separated on 1% agarose gels consisting of agarose IEF and ampholines with a range of 5.07.0 on a Phast-System (Amersham Biosciences, Orsay, France). The focused transferrin isoforms were visualized by immunofixation with a rabbit anti-human transferrin IgG (Dako, Trappes, France) applied directly on the gel for 15 min to precipitate the transferrin isoforms inside the gel. Proteins not bound by the antibody were washed out, the gel was stained with Coomassie blue R 250, and images were acquired using a charged coupled device (CCD) camera.
Purification of transferrin
Human serum (1.52 ml) was subjected to an affinity column made of rabbit anti-human transferrin IgG (Dako) coupled to activated Sepharose (Aminolink, Pierce, Rockford, IL) (Coddeville et al., 1998). Purification of transferrin was checked by SDSPAGE using 7.5% acrylamide gels (Laemmli, 1970
).
Ion-exchange purification of transferrin glycovariants
The transferrin glycovariants were separated on a Mono Q HR 5/5 column (Amersham Biosciences) by a NaCl gradient in Bis-Tris buffer 20 mM, pH 6.2 (Jeppsson et al., 1993). Each fraction was collected, dialyzed against water, and lyophilized. Protein content of each fraction was evaluated with bicinchoninic acid (BCA protein assay reagent, Pierce, Rockford, IL) (Smith et al., 1985
).
Release and purification of the transferrin glycans
Transferrin glycovariants (0.150.3 mg) were denatured at 100°C in the presence of SDS and ß-mercaptoethanol. After addition of nonidet-P 40, PNGase-F (New England Biolabs, Herts, UK) was added (1 µl) (500,000 U/ml). After 2 h, 1 µl additional enzyme was added and left overnight.
The oligosaccharides were purified by solid phase-extraction on a graphitized carbon adsorbant (Alltech Associates, Templemars, France); the neutral and the sialylated oligosaccharides were eluted together with a solution of acetonitrile 25/water 75 containing trifluoroacetic acid (0.1%) (Packer et al., 1998).
Fluorescent carbohydrate electrophoresis
Oligosaccharides released by PNGase-F were labeled by reductive amination with ANTS (Molecular Probe, Leiden, Netherlands) using the protocol described by Jackson (1994). ANTS-labeled oligosaccharides were subjected to PAGE on an isocratic 30% polyacrylamide gel using the Tris-glycine discontinuous buffer (Laemmli, 1970
), except that the SDS and the ß-mercaptoethanol were omitted from all buffers. Images were acquired using a CCD camera.
Extraction and desalting of ANTS oligosaccharides from the gel
The excised gel piece was washed without shaking in 500 µl of water at 4°C for 30 min. The gel fragment was then removed and subsequently reincubated in 500 µl of water for 34 h at 4°C. During the second step, the derivatized glycans diffused out of the gel, and the efficiency of sugar extraction was directly checked using an UV light. The extracted oligosaccharides were lyophilized and further analyzed by MS.
Esterification of sialic acids. To stabilize the sialic acid moiety under MALDI MS conditions, the sialic acid residues of PNGase-F-released oligosaccharides (500 pmoles) were stabilized by methylesterification of their carboxylic group (Powell and Harvey, 1996).
Exoglycosidase digestions. Aliquots of methylesterified and unmodified N-glycans were subjected to exoglycosidase digestions onto the mass spectrometer target, as described by Colangelo and Orlando (1999). Briefly, 1 µl of oligosaccharide samples (50 pmoles) was incubated on the target with 800 µU (0.25 µl) of neuraminidase or 56 µU (0.25 µl) of ß1-4 galactosidase for 10 and 20 min with both enzymes. The enzymatic digestions were stopped by acidification with 1 µl of MS matrix.
MS. All mass spectra were acquired on a Voyager Elite (DE-STR) reflectron time-of-flight (TOF) mass spectrometer (Perseptive Biosystems) equipped with a pulsed nitrogen laser (337 nm) and a gridless delayed extraction ion source. Samples were analyzed in delayed extraction mode using an accelerating voltage of 20 kV, a pulse delay time of 200 ns, and a grid voltage of 66%. Detector bias gating was used to reduce the ion current below masses of 500 Da. After external calibration, between 100 and 200 scans were averaged for each of the reflectron mode spectra shown.
For all measurements, the dried droplet preparation technique was employed. Native and methylesterified N-glycans were cocrystallized with DHB as matrix (10 mg/ml DHB in an acetonitrile/water solution [70:30] containing 0.1% trifluoroacetic acid). ANTS-labeled oligosaccharides were mixed with a freshly made 3-aminoquinoline matrix solution (10 mg/ml of 3-aminoquinoline in methanol/water [50/50] supplemented with 5 mM diammonium hydrogen citrate). ANTS-labeled glycans were detected as [M-H]- ions in linear negative mode, whereas the neutral sugars were observed as [M+Na]+ in positive reflectron mode.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: a-klein{at}chru-lille.fr
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Abbreviations |
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