Alcohol and Alcoholism Advance Access originally published online on October 3, 2005
Alcohol and Alcoholism 2006 41(1):18-23; doi:10.1093/alcalc/agh216
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DEFECTIVE GLYCOSYLATION OF CHOLESTERYL ESTER TRANSFER PROTEIN IN PLASMA FROM ALCOHOL ABUSERS
Department of Internal Medicine and Clinical Research Center, University of Oulu, Oulu, Finland
* Author to whom correspondence should be addressed at: Department of Internal Medicine, University of Oulu, PO Box 5000, 90014 Oulu, Finland. Tel: +358 8 573 6314; Fax +358 8 315 4543; E-mail: johanna.liinamaa{at}oulu.fi
(Received 16 November 2004; first review notified 20 January 2005; in revised form 13 June 2005; accepted 9 September 2005)
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ABSTRACT |
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Aims: Alcohol consumption reduces the carbohydrate content of some glycoproteins, e.g. carbohydrate-deficient transferrin. The aim of this study was to investigate if there is such an alcohol-induced glycosylation defect in plasma cholesteryl ester transfer protein (CETP). A defect in the posttranslational glycosylation of CETP may affect its structure and electrical charge and may therefore affect its function. CETP activity is low in alcohol abusers. Methods: We studied the effect of alcohol consumption on CETP properties in 10 alcohol abusers and 10 control subjects. CETP was partially purified from lipoprotein-free plasma by FPLC using a Phenyl-Sepharose column. Isoelectric focusing, polyacrylamide gel electrophoresis, and western blotting were performed for partially purified CETP. Results: CETP had a lower molecular weight in the alcohol abusers than in the controls (range 50.6–84.0 kDa in the alcohol abusers vs 51.3–85.0 kDa in the controls). CETP purified from alcohol abusers had a higher isoelectric point, indicating a lower negative charge on the surface of the protein than in the controls' CETP. A similar effect was observed when control CETP was incubated with neuraminidase, an enzyme which is known to remove sialic acid from glycoproteins. Conclusions: We conclude that CETP from alcohol abusers may have a glycosylation defect due to defective sialylation caused posttranslationally by alcohol itself or its metabolite acetaldehyde. The defective glycosylation of CETP associated with altered binding to lipoproteins may lead to the low CETP activity observed previously in alcoholic subjects.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cholesteryl ester transfer protein (CETP) is a plasma glycoprotein which transfers cholesteryl esters, triglycerides, and phospholipids between lipoproteins (Barter et al., 2003
). The molecular weight (MW) of purified plasma CETP has been reported to be 64–74 kDa (Albers et al., 1984; Hesler et al., 1987; Jarnagin et al., 1987) whereas the cDNA of CETP predicts a polypeptide of 53 kDa (Drayna et al., 1987). The difference between the deduced MW of CETP polypeptide and that of the mature protein is largely due to posttranslational modification in the liver including addition of asparagine N-linked carbohydrates (the chain terminated by N-acetyl neuraminic acid, also termed sialic acid) (Stevenson et al., 1993). Cholesteryl ester transfer protein has four potential N-linked glycosylation sites at positions 88, 240, 341, and 396 (Drayna et al., 1987), to which sialic acid residues are attached. In isoelectric focusing, CETP shows nine bands around an isoelectric point (pI) of 5.0 (Kato et al., 1989). Treatment of CETP with neuraminidase, an enzyme cleaving sialic acid residues from glycoproteins, results in a higher pI, indicating a lower negative charge of the carbohydrate-deficient CETP (Kato et al., 1989).
Chronic heavy alcohol consumption reduces the carbohydrate content of several N-linked glycoproteins (Gravel et al., 1996). Carbohydrate-deficient transferrin (CDT), a variant of serum transferrin is widely used as a marker of alcoholism (Stibler et al., 1981). CDT has a higher pI than transferrin due to lack of one or two carbohydrate chains (Landberg et al., 1995). The formation of CDT may be due to changes in several steps of glycoprotein turnover, such as decreased activity of sialyltransferase and other enzymes incorporating carbohydrates to glycoproteins (Stibler et al., 1991a; Ghosh et al., 1993), and increased activity of sialidase (Xin et al., 1995), an enzyme cleaving sialic acid residues. Reduced transferase activity, and therefore CDT formation, has been found to be due to enzyme inactivation by acetaldehyde rather than alcohol itself (Xin et al., 1995). Since CETP and transferrin are both glycoproteins with posttranslational attachment of sialic acid and other carbohydrates, similar changes as in the previously described CDT variant of transferrin might be found in CETP in the plasma of alcohol abusers.
The objective of this study was to explore whether alcohol abuse affects the carbohydrate content of plasma CETP. We hypothesized that defective glycosylation of CETP may reduce its MW and charge in isoelectric focusing. The decreased carbohydrate content of CETP might explain the decreased CETP activity observed previously in alcoholic subjects (Hannuksela et al., 1992), implying either decreased secretion or defective binding of CETP to lipoproteins in alcohol drinkers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Subjects
The study was carried out in a group of 11 male alcohol abusers and 10 controls. The alcohol abusers had been referred by general practitioners as inpatients to the Alcoholism Treatment Unit in Oulu for withdrawal therapy. This unit treats ambulatory patients seeking assistance for terminating their dependence on alcohol and who have no signs or symptoms of other diseases. Patients acutely ill with alcohol-related or other diseases are referred to other clinics. All the alcohol drinkers had been heavily drinking daily for at least 5 days before admission. The subjects were included if they were in a good nutritional state and did not have any clinical signs of kidney or heart dysfunction or, severe liver damage, such as spiders, jaundice or hepatomegaly. One subject was excluded on the basis of highly elevated liver enzymes (the exclusion criteria were alanine aminotransferase or aspartate aminotransferase values >160 U/l, alkaline phosphatase >750 U/l, or gamma glutamyltransferase >1200 U/l), leaving a total of 10 alcohol abusers. The liver enzymes were determined with the ECCLS system, except that alkaline phophatase was determined according to the recommendation of the Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology. The reference values were as follows: alanine aminotransferase and aspartate aminotransferase 10–50 U/l, alkaline phosphatase 60–250 U/l, or gamma glutamyltransferase 5–80 U/l. All the subjects gave their informed consent and the study design was approved by the local ethics committee.
The intake of alcohol among both the controls and the alcohol abusers was assessed through an extensive interview by a trained physician concerning the amount of beer, wine, and strong alcoholic beverages consumed during the previous two weeks. The alcohol intake was calculated and expressed in grams of pure alcohol per day. Information was also obtained on cigarette smoking, drug abuse, and past medical history. None of the alcohol abusers were skid-row alcoholics or dependent on narcotics. None of the alcohol abusers or the controls used prescription drugs or had diseases that might influence the lipid metabolism.
Ten healthy men from our laboratory staff served as control subjects. They were interviewed in the same manner as the alcohol abusers. Two of these men were teetotallers and the others reported occasional moderate alcohol intake.
Experimental design
Venous blood samples were collected into EDTA tubes after an overnight fast from both the alcohol abusers and the controls, those from the alcohol abusers being taken on the first day after their admission into the treatment unit. The samples for CETP activity, CDT determination, and gel filtration were stored at –70°C.
Polyclonal CETP antibody production in rabbits
A site-directed antibody against CETP peptide sequence was raised in rabbits. A C-terminal 31mer peptide (residues 446–476) of the CETP protein sequence was synthesized and purified in the Core Facilities of the Biocenter Oulu (Oulu, Finland). The CETP peptide (0.5 mg) was coupled with Traut's reagent (Pierce, Oud-Beijerland, The Netherlands) and then, through maleimido-N-benzoylsuccinamide, with 3 mg of keyhole limpet hemocyanin (Boehringer Mannheim Biochemica, Mannheim, Germany) as previously described (Francone et al., 1989). The complex was mixed with an equal volume of Freund's complete adjuvant (Sigma, St.Louis, MO) and injected into two rabbits in four equal portions 3 weeks apart. IgG class antibodies were purified by affinity chromatography using a HiTrap Protein A column (Pharmacia Biotech, Uppsala, Sweden).
Purification of plasma CETP
The d > 1.21 g/ml fraction was isolated from 120 ml of plasma from the control subjects and the alcohol abusers by ultracentrifugation in a Beckman Ti 50.2 rotor at 219 000 g and +15°C for 55 h, and applied to a HiLoad Phenyl-Sepharose 26/10 column (Pharmacia Biotech) as described earlier (Lagrost et al., 1994) using ÄKTA 10 FPLC equipment (Pharmacia Biotech). Hydrophobic proteins, including CETP, were eluted with distilled water (20 ml, 2 mg/ml of protein) and the fractions were used for isoelectric focusing. The purification of CETP was 
700-fold by this method.
SDS–PAGE
SDS–PAGE was carried out using 12% acrylamide separating gel in a vertical slab minigel apparatus (Bio-Rad, Hercules, CA). Plasma samples (1.7–4.0 µl, diluted 1:10, corresponding to CETP activity of 21.7 pmol/h) were applied to the gel. Some experiments were performed with equal volumes of plasma for all samples (4.0 µl, diluted 1:10). The proteins were electrophoretically transferred onto a nitro-cellulose membrane (Amersham, Buckinghamshire, UK) and then detected by western blotting. This was performed by first blocking the membrane for 1 h with 10% powdered milk in a solution of Tris-buffered saline and Tween (TBS–T) containing 20 mmol/l Tris, 137 mmol/l NaCl, 1 mmol/l HCl, 0.01% Tween, pH 7.6. Rabbit polyclonal CETP antibody was added to the solution at a concentration of 1:100 and incubated for 1 h. The membrane was then washed three times with TBS–T before incubation for 1 h with horseradish peroxidase-linked donkey anti-rabbit immunoglobulin (Amersham), which was diluted 1:3000 with 5% powdered milk in TBS–T solution. After washing as mentioned above, the membrane was treated with enhanced chemiluminescence reagents (Amersham) for 1 min before exposure to Kodak BioMax film (Eastman Kodak, Rochester, NY).
Isoelectric focusing
Native isoelectric focusing was performed by mini-gel using a 6% total/0.16% cross-linker acrylamide (Bio-Rad) containing 5% Ampholine 4-6 (Pharmacia Biotech). Samples (5–20 µl of elution fraction of Phenyl-Sepharose chromatography) were diluted 1:2 with sample buffer containing 60% glycerol, 20% Ampholine 4-6, 1% sucrose, and 0.01% bromophenol blue. The catholyte was 0.4% ethylenediamine (Fluka Chemie, Buchs, Germany) and anolyte 0.2% sulphuric acid (Merck, Darmstadt, Germany). Isoelectric focusing was performed at a constant voltage of 100 V for 1 h, 150 V for 30 min, and 200 V for 2.5 h. Proteins were then transferred electrophoretically in 0.7% acetic acid onto nitro-cellulose membrane and the membrane was blocked for 1 h with 10% powdered milk in a solution of TBS–T containing 20 mmol/l Tris, 137 mmol/l NaCl, 1 mmol/l HCl, 0.01% Tween, pH 7.6. Mouse monoclonal TP-2 antibody (a kind gift of Dr Yves L. Marcel, Ottawa, Canada) was diluted 1:1500 and incubated for 1 h. The membrane was then washed three times with TBS–T before incubation for 1 h with horseradish peroxidase-linked donkey anti-rabbit immunoglobulin (Amersham), which was diluted 1:3000 with 5% powdered milk in TBS–T solution. After washing as mentioned above, the membrane was treated with enhanced chemiluminescence reagents (Amersham) for 1 min before exposure to Kodak BioMax film (Eastman Kodak, Rochester, NY, USA). The western blotting films from SDS–PAGE and isoelectric focusing were quantified with QuantiScan software (Biosoft, Cambridge, UK), and the MWs and pIs were determined as a mean value of the area under the curve of the quantitation.
Neuraminidase treatment was performed on a CETP sample purified from a control subject to be used as a desialylated control in isoelectric focusing. The elution fraction from Phenyl-Sepharose chromatography (200 µl) was mixed with 20, 10, and 5 µl of neuraminidase from Clostridium Perfringens attached to beaded agarose (corresponding to 8.8, 4.4, and 2.2 ng of neuraminidase, respectively) (Sigma). The total volume of mixture was brought to 400 µl by adding 4.0 µl of 1 mol/l sodium phosphate buffer, pH 7.4, and distilled water to a final concentration of 10 mmol/l sodium phosphate buffer. The mixtures were incubated at +37°C for 15, 5, and 2 min, after which neuraminidase was removed by centrifuging at 1500 g for 2 min, and the supernatant was used in analysis as a desialylated control.
Chemical analyses
The plasma cholesterol, free cholesterol, triglyceride, and phospholipid concentrations were determined by enzymatic colorimetric methods using a Kone Specific Selective Chemistry Analyzer (Kone, Espoo, Finland) and kits of Boehringer Diagnostica catalogue nos. 236691, 310328, and 701912 (Boehringer Mannheim), and Wako no. 990-54009 (Wako Chemicals, Neuss, Germany), respectively. Using a CDTect Radioimmunoassay kit (Pharmacia, Uppsala, Sweden, not available on the market anymore) (Stibler et al., 1991b) the CDT was measured after dialysing the plasma samples against 0.15 mol/l NaCl.
Determination of CETP activity
Activity of CETP was determined in (VLDL + LDL)-free plasma as described earlier (Hannuksela et al., 1992) by detecting the exchange of radioactive cholesteryl esters between labelled LDL and unlabelled HDL, both isolated from the control subjects. This method reflects the CETP protein concentration (Hannuksela et al., 1992). The intra-assay coefficient of variation in plasma CETP activity was 6% and the inter-assay variation was also 6%.
Statistical methods
The results are given as means and standard deviation (SD). The differences between the means were calculated with Students t-test using the SPSS version 9.0 (Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical characteristics
The clinical characteristics are shown in Table 1. The mean daily alcohol consumption was 155 g in the group of alcohol abusers and 12 g in the control group. Mean age and body mass index did not differ significantly between the alcohol abusers and the controls, but liver enzymes were slightly elevated in the alcohol abusers. In the alcohol abusers CETP activity was 25% lower (P < 0.001) than in the controls. The CDT values, as well as plasma HDL cholesterol were higher in the alcohol abusers than in the controls (Table 1).
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Molecular weight of CETP in the alcohol abusers and the controls
To determine the MWs of CETP in the alcohol abusers and the controls, plasma was applied to the SDS–PAGE gels and, after electrophoresis, western blotting was performed with rabbit polyclonal anti-CETP IgG. A typical pattern of SDS–PAGE is shown in Fig. 1A. Typically for glycoproteins, CETP was observed as a broad band both in the alcohol abusers and the controls. We have calculated mean of the area under the curve from densitometric scanning of the gels and gained mean MW and mean pI for the alcohol abusers and the controls. The mean MW was 63.800 kDa (95% confidence interval 62.471–65.160) for the alcohol abusers and 65.200 kDa (95% confidence interval 63.434–66.986) for the controls, but the difference was not statistically significant (P = 0.17692, independent samples T-test). However, the band was located at slightly lower MW in the alcohol abusers than in the controls (range 50.6–84.0 kDa in the alcohol abusers vs 51.3–85.0 kDa in the controls, n = 10 for both groups), the CETP concentrations in the samples were equal (Fig. 1B). Similar results were observed when equal volumes were applied on the SDS–PAGE gels.
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Isoelectric point of CETP in the alcohol abusers and the controls
Native isoelectric focusing was performed on partially purified CETP to detect possible changes in the charge of CETP. The results from the isoelectric focusing and western blotting are shown in Fig. 2A. CETP was observed as either eight or nine bands between pI 4.80 and 5.30 in the alcohol abusers, and as eight bands between 4.78 and 5.20 in the controls. The results from the scanning and quantitation of isoelectric focusing are shown in Fig. 2B. The pI of CETP was 5.015 for the alcohol abusers and 4.985 for the controls, determined as a mean value of the area under the curve of the quantitation curve. The most acidic two or three bands with pI
4.8, were less stained in the alcohol abusers than in the controls, indicating a loss of sialic acid in highly sialylated CETP forms (Fig. 2B, indicated with arrows). The differences in pI were not statistically significant nor related to the amount of alcohol intake among the alcohol abusers. The pI of CETP was not correlated to plasma CETP activity or CDT concentration.
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Neuraminidase treatment was performed on the CETP samples purified from one control subject to be used as a desialylated control in isoelectric focusing (see Fig. 2A, lanes 5 and 6). A brief neuraminidase treatment (for 2 min) resulted in the appearance of a new band at pI 5.2, and the distribution of bands was similar to that observed in the alcohol abusers, although the bands were weaker in the alcohol abusers. Furthermore, a longer treatment (for 15 min) with a stronger neuraminidase concentration led to the formation of multiple bands in a higher pI region (from pI 5.2–5.4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The current study provides evidence of defective glycosylation of CETP in plasma from alcohol abusers by showing lower MW and lower negative charge of CETP compared with the controls. The current results may, at least in part, explain the low CETP activity and mass in the alcohol abusers (Hannuksela et al., 1996
) and contribute to our previous results of altered cholesteryl ester transfer observed in the alcohol abusers (Liinamaa et al., 1997).
In the current study, we describe a reduction in the MW of CETP from alcohol abusers. Some previous studies report MW of 74 kDa of highly purified human CETP (Jarnagin et al., 1987). However, several other studies, where CETP was less purified, indicated CETP MW of 58–66 kDa (Ihm et al., 1982; Tollefson and Albers, 1986; Busch et al., 1987). The current results are comparable with the latter studies showing lower MW of partially purified CETP and the difference between the MW values of alcoholic and control CETP, 2 kDa, may reflect the existence of a poorly glycosylated form (lack of <10 sugars, i.e. approximately one carbohydrate chain) of CETP in the plasma of alcohol abusers.
Sialic acid is the only charged carbohydrate in glycoproteins. One molecule of CETP contains 6.3 molecules of sialic acid, ranging from 3 to 11 sialic acid residues per CETP molecule, each contributing to one band in isoelectric focusing (Kato et al., 1989). In the current study, the reduction in the charge of CETP in the alcohol abusers shown as one or two extra bands in isoelectric focusing corresponds to the existence of a CETP isoform lacking at least one sialic acid. The weaker intensities in lower pI bands reflect decreased concentrations of highly sialylated CETP. The results thus suggest that CETP in the alcohol abusers might have a glycosylation defect that reduces the charge of CETP.
The defectively glycosylated form of CETP is analogous to CDT, the carbohydrate-poor isoform of serum transferrin. However, the glycosylation defect in CETP was not correlated with CDT concentration in the alcohol abusers. Due to differences in the carbohydrate content of CETP and transferrin (Marz et al., 1982; Kato et al., 1989), the alcohol-induced glycosylation defect may vary, thereby affecting the secretion of the proteins. Furthermore, the defectively glycosylated CETP, especially when attached to VLDL as previously shown (Hannuksela et al., 1996), may be catabolized more rapidly than CDT. A previous study suggests that the CDT variant comprises a maximum of 21% of total transferrin (Stibler et al., 1980). Our results suggest that the percentage of the defectively glycosylated form of CETP may be lower than that for CDT and vary considerably between the individuals, partly explaining the lack of correlation between defectively glycosylated CETP and CDT. The reason for the interindividual variation is not known. We provide here evidence of defectively glycosylated CETP, but more precise methods, such as described by Varki (1991), for analysing purified CETP are needed to fully elucidate the exact alcohol-induced defect in glycosylation of CETP and its role in the function of CETP.
We have previously shown that the activity and concentration of CETP are low in alcohol abusers (Hannuksela et al., 1992) and the cholesteryl ester transfer is altered in alcohol abusers compared with controls (Liinamaa et al., 1997), a phenomenon which is affected by alcohol-induced differences in lipoprotein concentration and composition. Lipoprotein composition strongly influences the binding of CETP to lipoproteins, which is dependent on alterations in the charge characteristics of lipoproteins (Bruce et al., 1995). Both excessive and insufficient lipoprotein–CETP interactions reduce the cholesteryl ester transfer (Morton and Greene, 2003). Moreover, changes in the charge of CETP may affect lipoprotein–CETP interactions and the function of CETP. The binding of CETP to lipoproteins and the catalysis of transfer are suggested to locate in the C-terminal region of CETP (Au-Young and Fielding, 1992), and charge alterations in its proximity may affect the function of CETP. The present results add an interesting viewpoint to our previous results on altered cholesteryl ester transfer in alcohol abusers, since defectively glycosylated CETP may, due to changes in its conformation and charge, be differently attached to lipoproteins, thereby influencing the function of CETP in cholesteryl ester transfer (Liinamaa et al., 1997).
In conclusion, CETP purified from alcohol abusers shows a glycosylation defect that may be due to defective sialylation caused posttranslationally by alcohol itself or its metabolite acetaldehyde. The defective sialylation of CETP could explain the low plasma CETP activity and the altered cholesteryl ester transfer observed previously in alcoholic subjects, possibly by defective binding of CETP to lipoproteins in alcohol drinkers.
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ACKNOWLEDGEMENTS |
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We wish to thank the staff of the Kiviharju Alcoholism Treatment Unit for their co-operation. The skillful technical assistance of the technicians in the Department of Internal Medicine at the University of Oulu is greatly appreciated. This work was supported by the Finnish Foundation of Alcohol Studies, the Finnish Foundation for Cardiovascular Research, the Academy of Finland, Astra Finland, and the Finnish Medical Foundation. The TP-2 antibody was a kind gift of Dr Yves L. Marcel, Lipoprotein & Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa Civic Hospital, Ottawa, Canada.
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