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Clinical Chemistry 45: 1408-1413, 1999;
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(Clinical Chemistry. 1999;45:1408-1413.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Microheterogeneity of Serum Glycoproteins in Patients with Chronic Alcohol Abuse Compared with Carbohydrate-deficient Glycoprotein Syndrome Type I

Hugues Henry1,a, Florian Froehlich3, Renaud Perret3, Jean-Daniel Tissot4, Barbara Eilers-Messerli1, Daniel Lavanchy2, Carlo Dionisi-Vici5, Jean-Jacques Gonvers3 and Claude Bachmann1

1 Central Clinical Chemistry Laboratory and
2 Division of Immunology and Allergology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.

3 Division of Gastroenterology, Policlinique Médicale Universitaire, Lausanne, Switzerland.

4 Red Cross Transfusion Center, Lausanne, Switzerland.

5 Department of Metabolism, Bambino Gesu Hospital, Rome, Italy.
a Address correspondence to this author at: Laboratoire Central de Chimie Clinique, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland. Fax 41-21-3144288; e-mail Hugues.Henry{at}chuv.hospvd.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Chronic alcohol abuse alters the normal N-glycosylation of transferrin, producing the carbohydrate-deficient transferrin isoforms. This alteration could be similar to that present in patients with carbohydrate-deficient glycoprotein syndrome type 1 (CDG1). We thus compared the alterations of N-glycans present in patients with alcoholism and patients with CDG1.

Methods: The N-glycans of serum glycoproteins were compared in sera of patients with alcoholism, patients with CDG1, and controls by two-dimensional electrophoresis, neuraminidase, peptide:N-glycosidase F, and endoglycosidase F2 treatments. A specific antibody directed against the amino acid sequence surrounding the N-432 N-glycosylation site of transferrin was prepared (SZ-350 antibody).

Results: In patients with alcoholism, the abnormal transferrin and {alpha}1-antitrypsin isoforms were devoid of a variable number of entire N-glycan moieties and were identical with those present in CDG1. In the serum of patients with alcoholism, this finding was less pronounced than in CDG1. In contrast to CDG1, there was no decrease in clusterin or serum amyloid P in patients with alcoholism. The SZ-350 antibody recognized only transferrin isoforms with one or no N-glycan moieties.

Conclusion: Antibodies directed against specific N-glycosylation sites of glycoproteins could be useful for developing more specific immunochemical tests for the diagnosis of chronic alcohol abuse.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic alcohol abuse is known to alter the core glycosylation of serum transferrin, producing the carbohydrate-deficient transferrin (CDT)1 isoforms (1)(2)(3). These alterations have been described as related to a reduced number of N-glycans on transferrin (4)(5).

Extremely high CDT values are also present in patients with carbohydrate-deficient glycoprotein syndrome type 1 (CDG1) disorders (6). CDG1 disorders are autosomal recessive and multisystemic disorders with neonatal or infantile presentation affecting mainly the central and peripheral nervous system (6). The disease is attributable to a decrease of guanosine diphosphate (GDP)-mannose availability secondary to either phosphomannomutase (EC 5.4.2.8) (7)(8) or phosphomannose isomerase deficiency (EC 5.3.1.8) (9).

The biochemical comparison of the alteration of glycoproteins in patients with chronic alcohol abuse with the alterations in CDG1 patients could help to better define the underlying mechanism in alcoholism. Therefore, we studied the microheterogeneity of glycoproteins by using high-resolution two-dimensional polyacrylamide electrophoresis (2D-PAGE) of serum from patients with chronic alcoholism and compared them to the patterns in CDG1 patients. The use of new antibodies, which are specifically directed against the N-glycan binding site localized on N-432 of transferrin allowed us to immunodetect transferrin devoid of glycans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patient samples
Serum from patients with chronic alcohol abuse (n = 26) were collected in the Division of Gastroenterology (Policlinique Médicale Universitaire, Lausanne); total CDT concentrations were obtained using the commercially available CDTectTM (Amersham-Pharmacia-Biotech). For comparison, sera from 12 patients with CDG1 were analyzed (10). Control sera (n = 10) were obtained from blood donors of the Red Cross Transfusion Center (Lausanne) and from teetotalers.

2d-page
Serum proteins were subjected to 2D-PAGE as described previously (10)(11)(12). Briefly, in the first dimension, isoelectric focusing was performed using Immobiline strips (180 x 3 mm) with a nonlinear pH gradient from pH 3 to pH 10 (Amersham-Pharmacia-Biotech). In the second dimension, sodium dodecyl sulfate (SDS)-PAGE was performed on 200 x 170 x 1.5 mm gels containing 90–160 g/L total acrylamide and 26 mg/g cross-linker. The proteins were either silver stained (13) or immunodetected with monospecific antibodies directed against human transferrin and {alpha}1-antitrypsin (Dako) (10).

specific antibodies for the n-432 n-glycosylation site of transferrin
Rabbit antibodies were raised against the peptide NH2-LAENYNKSDNC-COOH (custom made by Eurogentec, B-Seraing). This peptide is encoded by amino acid sequence 427–437 of human transferrin (accession number P02787; SwissProt database) and contains in its center the N-432 N-glycan binding site of the protein surrounded by 10 coding amino acids. The synthetic peptide was linked to keyhole limpet hemocyanin through the sulfhydryl group of its C-terminal cysteine, and the products were dialyzed against phosphate-buffered saline (14). Two rabbits were injected subcutaneously with the dialysate emulsified in complete Freund's adjuvant. They were boosted 2, 5, and 9 weeks later with an emulsion prepared in incomplete Freund's adjuvant and bled at week 13. IgGs were precipitated in 500 mg/g ammonium sulfate and dissolved in phosphate-buffered saline at a final protein concentration of 40 g/L. The preparation of antibody that showed the highest binding for the antigenic peptide (SZ-350 antibody) was selected for further experiments.

For Western blots, SZ-350 antibody was diluted 1000-fold in blocking buffer (50 g/L dry milk and 3 mL/L Tween 20 in phosphate-buffered saline) followed by peroxidase-linked second antibodies (Bio-Rad) diluted 4000-fold in blocking buffer. The immunodetected proteins were revealed by chemiluminescence (ECL; Amersham-Pharmacia) with 10-s exposure. The radiographs (Kodak X-OMAT AR; Integra Biosciences) were scanned with a laser densitometer (Molecular Dynamics, Bucher) and processed by image analysis (ImageQuant Ver. 3.3 software; Molecular Dynamics, Bucher).

The specificity of the targeted SZ-350 antibody was compared to the specificities of polyclonal antibodies directed against several transferrin epitopes (Dako), using sera from CDG1 patients. The total transferrin concentrations were measured by immunoturbidimetry (Roche Diagnostics), and the relative abundance of each transferrin isoform was determined by Western blot using polyclonal antibodies (Dako). The content of transferrin in each isoform was calculated as the result of the total transferrin multiplied by the relative abundance of the isoforms.

neuraminidase, endoglycosidase f2, and peptide:n-glycosidase f treatments
The glycoproteins were treated with neuraminidase from Clostridium perfringens (Roche Molecular Biochemicals), endoglycosidase F2, and peptide:N-glycosidase F from Flavobacterium meningosepticum (Roche Molecular Biochemicals) as described previously (10)(15). At the end of the incubations, the proteins were separated by SDS-PAGE on 200 x 170 x 1 mm gels containing 90 g/L total acrylamide and 26 mg/g cross-linker and then immunodetected as described previously using 2D-PAGE and SZ-350 antibody.

statistical analysis
The Passing-Bablock (16) test was used for regression, and the Mann–Whitney U-test was used for group comparisons. All tests were performed with the Analyze-It software package (Analyze-it Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
microheterogeneity of serum glycoprotein isoforms
Serum proteins from patients with chronic alcohol abuse (n = 10), from patients with CDG1 (n = 12), and from controls (n = 10) were separated by 2D-PAGE and then either silver stained or immunodetected. Serum glycoproteins from patients with alcoholism presented abnormal isoforms of transferrin, {alpha}1-antitrypsin, and haptoglobin ß chains with decreased masses and charge alterations when silver stained (Fig. 1 A, indicated by arrowheads). In contrast to CDG1 (10), there was no reduction of clusterin and serum amyloid P in patients with alcoholism.



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  		Figure 1. High-resolution 2D-PAGE separation of serum proteins.

Proteins were separated in the first dimension (abscissa) by isoelectric focusing electrophoresis and in the second dimension (ordinate) by SDS-PAGE as described in Materials and Methods. (A), whole gel showing the pathognomonic pattern of affected serum glycoproteins from a patient with chronic alcohol abuse. Proteins were silver stained. (B), enlargements of gels of immunodetected transferrin and {alpha}1-antitrypsin from a control and patients with alcoholism or CDG1. Trains of carbohydrate-deficient glycoproteins are marked with arrowheads.

The electrophoretic patterns of immunodetected transferrin and {alpha}1-antitrypsin isoforms from one representative patient with chronic alcohol abuse, one patient with CDG1, and a control are shown in Fig. 1BUp .

In controls, transferrin was present as a major row of up to six spots with the same mass of 77 kDa but with pI values of 6.15–6.44. However, in patients with alcoholism and patients with CDG1, two additional rows of spots with decreased masses and with cathodal shift (loss of negative charges) were found. The first additional row contained up to five spots with a molecular mass of 74 kDa and pI values of 6.39–6.58; the second additional row contained up to three spots with a molecular mass of 70 kDa and pI values of 6.49–6.58.

{alpha}1-Antitrypsin isoforms were present in controls as a row of closely stacked spots with masses of 56 to 54 kDa (mean, 55 kDa) and pI values of 4.91–5.10. In patients with alcoholism as well as in patients with CDG1, additional and similar spots with decreased masses of 52 and 49 kDa and pI values of 5.10–5.30 were observed.

neuraminidase and peptide:n-glycosidase f treatments
To characterize the N-glycosylation of the abnormal isoforms, the glycoproteins were subjected to neuraminidase and peptide:N-glycosidase F treatments.

Neuraminidase treatment leads to the removal of all the terminal sialic acids of the N-glycans. Except for the abnormal band (70 kDa) of transferrin present in patients with alcoholism and patients with CDG1, all other normal and abnormal bands of isoforms of transferrin or of {alpha}1-antitrypsin were shifted to lower masses with a molecular mass difference of 500-1200 Da (detection limit, 500 Da), which is compatible with the loss of one to four sialic acids (molecular mass, 309 Da; Fig. 2 , lane 2 of TRF and AAT). The 70-kDa transferrin isoform can be considered as the asialo-transferrin form.



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  		Figure 2. Immunoblots of serum transferrin (TRF) and {alpha}1-antitrypsin (AAT) isoforms separated by SDS-PAGE from a control, a patient with chronic alcohol abuse, and a patient with CDG1.

A volume of 10 µL containing 0.125 µL of serum was applied to each lane; the polyclonal antibodies were directed against several epitopes. The glycoproteins were either not treated (lane 1) or treated with neuraminidase (lane 2) or peptide:N-glycosidase F (lane 3). The right axis (N-glycans) indicates the number of glycans still linked to the surface of the glycoproteins.

Peptide:N-glycosidase F treatment produced isoforms whose masses varied as a function of the number of N-glycans still linked to the proteins (Fig. 2Up , lane 3 of TRF and AAT); transferrin that usually contains two N-linked glycans generated three bands corresponding to isoforms with zero, one, and two N-glycans; and {alpha}1-antitrypsin (three N-glycans) generated four bands. In controls, patients with alcoholism, and patients with CDG1, similar bands of the generated fragments had identical electrophoretic mobility.

quantitative assay by sds-page and immunoblot
The intensities of the normal and abnormal bands of the transferrin and {alpha}1-antitrypsin isoforms in controls, patients with alcoholism, and CDG1 patients are presented in Table 1 . In patients with alcoholism, the relative abundance of the serum isoforms with a reduced number of N-glycans on transferrin and {alpha}1-antitrypsin was less extensive and more variable than in the sera of CDG1 patients. The abnormal transferrin isoform of 74 kDa, which carries only one N-glycan, in heavy drinkers is more prevalent than the isoforms with 70 kDa (asialo-transferrin).


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  		Table 1. Relative distribution of normal and abnormal bands of isoforms of transferrin and {alpha}1-antitrypsin.1

A pattern comparison of the transferrin isoforms by Western blot, using either commercially available antibodies (directed against several epitopes) or the SZ-350 antibody targeted to the N-432 epitope is shown in Fig. 3 A. In both controls and patients, SZ-350 did not react with the normal 77-kDa transferrin isoform with two linked N-glycans. Furthermore, in patients with chronic alcohol abuse and in patients with CDG1, the SZ-350 antibody bound only to the 74- and 70-kDa CDT isoforms.



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  		Figure 3. Comparison of the specificity of antibodies.

Transferrin isoforms by antibody directed against multiple epitopes [polyclonal anti-human transferrin antibody from Dako; anti-TRF (Dako)] and by the epitope-targeted SZ-350. (A), serum from a representative control (lane 1), from an patient with alcoholism (lane 2), and from a CDG1 patient (lane 3). (B), serum from a control (lane 1) and control serum digested with neuraminidase, endoglycosidase F2, or peptide:N-glycosidase F (lanes 2–4, respectively).

After treatment of normal serum transferrin with neuraminidase, endoglycosidase F2 (which removes all the carbohydrates except for the linked N-acetylglucosamine) or peptide:N-glycosidase F, SZ-350 antibody recognized only the band generated by peptide:N-glycosidase F (Fig. 3BUp ).

Densitometric quantification of the immunodetected 74- and 70-kDa transferrin isoforms by SZ-350 was linear for 0–12.5 µmol/L incompletely glycosylated transferrin (sum of nonglycosylated and partially glycosylated transferrin) when the Dako antibody was used (Fig. 4 A). The slope for the 70-kDa transferrin isoform was twice that of the 74-kDa isoform.



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  		Figure 4. Densitometric quantification of the immunodetected 74- and 70-kDa transferrin isoforms.

(A), comparison of SZ-350 antibody response with the sum of nonglycosylated and partially glycosylated transferrin isoforms with the Dako antibodies. (B), 74- and 70-kDa transferrin isoforms in patients with chronic alcohol abuse vs their CDTect values. The transferrin concentrations of the SZ-350 antibody are derived from the slope shown in panel A.

Serum specimens from 26 patients with alcoholism were evaluated by the CDTect test and the SZ-350 Western blot methods (Fig. 4BUp ). There was a positive correlation between CDTect values and 74-kDa (r2 = 0.51; P <0.001) and 70-kDa (r2 = 0.68; P <0.001) transferrin isoforms. This last isoform was not detected in patients with alcoholism who had CDT values <40 units/L. The intercept of the curve for immunodetected 74-kDa transferrin (25 units/L) is close to the cutoff values of CDT (<=20 units/L for men and <=26 units/L for women).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
n-glycan deficiency in patients with chronic alcohol abuse
In patients with chronic alcohol abuse and with CDG1, 2D-PAGE separation and immunodetection of the abnormal transferrin and {alpha}1-antitrypsin isoforms showed similar abnormalities with respect to decreased mass and charge. In addition, neuraminidase and peptide:N-glycosidase F treatments indicated that homologous species of N-glycans with sialic acid were still linked to the transferrin and {alpha}1-antitrypsin of the abnormal isoforms from patients with alcoholism and that these abnormal isoforms were devoid of a variable number of entire N-glycan moieties in patients with alcoholism or CDG1.

Our data thus indicate that patients with alcoholism have alterations of their transferrin and {alpha}1-antitrypsin that are analogous to those in patients with CDG1. However patients with alcoholism and patients with CDG1 differed in the number of glycan moieties left on the protein.

Previous reports suspected the presence of intermediate or truncated forms of N-linked glycans on glycoproteins in patients with chronic alcohol abuse (3)(17), and recently, several authors (4)(5) have demonstrated that patients with alcohol abuse are lacking N-glycans on transferrin. The results presented here indicate that complete N-glycans are still present on the serum glycoproteins. Furthermore, both transferrin and {alpha}1-antitrypsin are defective in N-glycan addition as well.

specific antibodies directed against cdt
The SZ-350 antibodies we used recognize the N-432 N-glycan binding site of transferrin only if the protein lacks its entire N-glycan moiety. The antibodies bind specifically to transferrin isoforms lacking one or two entire N-glycans. However, they do not bind either to the normal transferrin isoform with two N-glycans or to transferrin with N-glycans truncated by neuraminidase and by endoglycosidase F2. These data suggest that the steric hindrance of the entire or truncated N-glycan on transferrin impedes its further binding.

Because peptide:N-glycosidase F converts asparagine (N) to aspartate (D), SZ-350 recognizes the peptide sequence with either N-432 or D-432. In addition, the binding of the SZ-350 antibodies appears to be highly specific for the abnormal of 74- and 70-kDa transferrin isoforms present in patients with chronic alcohol abuse or with CDG1. The immunodetected response for the 70-kDa isoform was twice that of the 74-kDa isoform. This is compatible with the observation (18) that in patients with CDG1, loss of the N-glycosylation for 74-kDa transferrin occurs randomly at each glycosylation sites of the protein, i.e., that 50% of the 74 kDa isoform has one N-glycan at N-432.

Patients with CDG1 are deficient either in phosphomannose isomerase (9) or phosphomannomutase (7)(8)(19). Both of these defects alter the metabolism of GDP-mannose in cells and lead to a decrease of GDP-mannose for N-glycan synthesis, which affects the early steps of N-glycan transfer in the endoplasmic reticulum. Our data suggest that in addition to a decrease of galactosyl and sialyltransferase activities (20)(21)(22) chronic alcoholism in adults could affect the initial mannose-dependent steps of N-glycan synthesis.

The observation that, in contrast to patients with CDG1, there was no decrease in clusterin or amyloid P in the serum of patients with alcoholism needs further investigation.

CDT is used as a marker of chronic alcohol consumption (23)(24). The widely used commercial assay CDTect separates the transferrin isoforms by anion-exchange chromatography using microcolumns (25) before immunoassay of the fraction containing several isoforms; it is based on charge alterations of transferrin. However, the method does not allow a clear-cut separation between normal and abnormal isoforms (26). In addition, the method leads to false-positive results with the genetic variants B and D of transferrin (27). SZ-350 antibodies have the potential to provide a new immunochemical tool for CDT determination that is independent of the transferrin genotype and would eliminate the initial separation step of anion-exchange chromatography.


   Acknowledgments
 
We thank H. Decrey, MD, and B. Yersin, MD, for providing sera from patients with chronic alcohol abuse.


   Footnotes
 
1 Nonstandard abbreviations: CDT, carbohydrate-deficient transferrin; CDG1, carbohydrate-deficient glycoprotein syndrome type 1; GDP, guanosine diphosphate; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; and SDS, sodium dodecyl sulfate.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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F. J. Legros, V. Nuyens, E. Minet, P. Emonts, K. Z. Boudjeltia, A. Courbe, J.-L. Ruelle, J. Colicis, F. de L'Escaille, and J.-P. Henry
Carbohydrate-deficient Transferrin Isoforms Measured by Capillary Zone Electrophoresis for Detection of Alcohol Abuse
Clin. Chem., December 1, 2002; 48(12): 2177 - 2186.
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 Mol. Cell. ProteomicsHome page
N. L. Anderson and N. G. Anderson
The Human Plasma Proteome: History, Character, and Diagnostic Prospects
Mol. Cell. Proteomics, November 1, 2002; 1(11): 845 - 867.
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 GlycobiologyHome page
H. H. Freeze
Update and perspectives on congenital disorders of glycosylation
Glycobiology, December 1, 2001; 11(12): 129R - 143R.
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 Alcohol AlcoholHome page
A. Helander, M. Fors, and B. Zakrisson
STUDY OF AXIS-SHIELD NEW %CDT IMMUNOASSAY FOR QUANTIFICATION OF CARBOHYDRATE-DEFICIENT TRANSFERRIN (CDT) IN SERUM
Alcohol Alcohol., September 1, 2001; 36(5): 406 - 412.
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A. Helander, G. Eriksson, H. Stibler, and J.-O. Jeppsson
Interference of Transferrin Isoform Types with Carbohydrate-deficient Transferrin Quantification in the Identification of Alcohol Abuse
Clin. Chem., July 1, 2001; 47(7): 1225 - 1233.
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 CVIHome page
M. A. Smith, S. K. Bains, J. C. Betts, E. H. S. Choy, and E. D. Zanders
Use of Two-Dimensional Gel Electrophoresis To Measure Changes in Synovial Fluid Proteins from Patients with Rheumatoid Arthritis Treated with Antibody to CD4
Clin. Vaccine Immunol., January 1, 2001; 8(1): 105 - 111.
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T. Arndt
Carbohydrate-deficient Transferrin as a Marker of Chronic Alcohol Abuse: A Critical Review of Preanalysis, Analysis, and Interpretation
Clin. Chem., January 1, 2001; 47(1): 13 - 27.
[Abstract] [Full Text] [PDF]

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L. Dibbelt
Does Trisialo-Transferrin Provide Valuable Information for the Laboratory Diagnosis of Chronically Increased Alcohol Consumption by Determination of Carbohydrate-deficient Transferrin?
Clin. Chem., August 1, 2000; 46(8): 1203 - 1205.
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E. Korvatska, H. Henry, Y. Mashima, M. Yamada, C. Bachmann, F. L. Munier, and D. F. Schorderet
Amyloid and Non-amyloid Forms of 5q31-linked Corneal Dystrophy Resulting from Kerato-epithelin Mutations at Arg-124 Are Associated with Abnormal Turnover of the Protein
J. Biol. Chem., April 6, 2000; 275(15): 11465 - 11469.
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R. Hackler, T. Arndt, A. Helwig-Rolig, J. Kropf, A. Steinmetz, and J. R. Schaefer
Investigation by Isoelectric Focusing of the Initial Carbohydrate-deficient Transferrin (CDT) and non-CDT Transferrin Isoform Fractionation Step Involved in Determination of CDT by the ChronAlcoI.D. Assay
Clin. Chem., April 1, 2000; 46(4): 483 - 492.
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G.-C. Wu, H.-L. Lai, Y.-W. Lin, Y.-T. Chu, and Y. Chern
N-Glycosylation and Residues Asn805 and Asn890 Are Involved in the Functional Properties of Type VI Adenylyl Cyclase
J. Biol. Chem., September 14, 2001; 276(38): 35450 - 35457.
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