Electrophoresis 199 I, 12, 59-63
Immunoblotting of apolipoprotein E
59
Winfried Marz Sabine Cezanne Werner GroB
Phenotyping of apolipoprotein E by immunoblotting in immobilized pH gradients
Gustav Embden-Centerof Biological Chemistry, J. W. Goethe-University, Frankfurt/Main
An immunoblotting method for the determination of apolipoprotein E (apoE) phenotypes has been developed. Delipidated plasma proteins are focused in an immobilized pH gradient, and transferred to polyvinylidene difluoride (PVDF) membranes. ApoE isomorphs are identified by immunoperoxidase staining. The method allows reproducible assignment of apoE phenotypes without isolation of triglyceride-rich lipoproteins. Only small amounts of serum are required. There are several important steps in the procedure: (i) delipidation is indispensable, (ii) carrier ampholytes have to be added to the gels and to the sample buffer, and, (iii) on immunostaining, polyvinylidene difluoride membranes provide an excellent signal-tobackground ratio.
1 Introduction Apolipoprotein E (apoE) is a well-characterized 34 kDa glycoprotein, associated with triglyceride-rich and high density lipoproteins [ 1-31. It is a ligand for low density lipoprotein (LDL) receptors and hepatic lipoprotein remnant receptors, and plays a key role in the catabolism of triglyceride-rich lipoproteins 14-61. Three alleles ( ~ 2~, 3~, 4at) the apoE locus give rise to three homozygous (E2/2, E3/3, and E4/4) and three heterozygous phenotypes (E3/2, E4/2, and E4/3). This polymorphism is due to cysteine-arginine interchanges at amino acid residues 1 12 and 158. ApoE4 contains arginine and E2 contains cysteine at both sites, whereas E3 has cysteine at position 112 and arginine at position 158 [71. Other rare alleles have also been reported [8- 141.ApoE2 is defective in binding to apoB, E receptors 115-171. Because the clearance of both chylomicron and very low density lipoprotein (VLDL) remnants depends on functional apoE, homozygotes for apoE2 accumulate remnant particles in their circulation. Concomitantly, the flux of dietary cholesterol into the liver is decreased and hepatic apoB,E receptors are up-regulated. Therefore, E2 homozygotes have low LDL cholesterol and apoB levels. However, in response to some additional genetic, metabolic or environmental factors, a small fraction of E2 homozygotes expresses type I11 hyperlipoproteinemia [4-6, 181. The ~4 allele exerts an opposite effect on lipoprotein levels: It is associated with high LDL cholesterol and apo B levels, and appears to predispose to premature atherosclerosis [4-6, 191. Conventionally, apoE phenotyping has been performed by isoelectric focusing (IEF) of delipidated triglyceride-rich lipoproteins 120-251. Immunoblotting of apoE after IEF in carrier ampholyte (CA)-based pH gradients has been reported [26-291. Moreover, it has been demonstrated that IEF of delipidated VLDL in immobilized pH gradients (IPG) affords improved apoE phenotyping [301. We have developed a method that combines the advantages of IEF in IPGs with thoseofimmunoblotting [3 I]. Here we report on the optimization of this procedure. Particular emphasis is placed on the methodical Correspondence: Prof. Dr. W. Grofi, Gustav Embden-Center of Biological Chemistry, Johann Wolfgang Goethe-University, Theodor Stern-Kai 7, D-6000 Frankfurt am Main, Germany Abbreviations: apo, apolipoprotein; CA, carrier ampholytes; HRP, horseradish peroxidase; IEF, isoelectric focusing; IPG, immobilized pH gradient; PVDF, polyvinylidene difluoride; Tris, tris (hydroxymethyl) aminomethane; VLDL, LDL, HDL, very low, low, and high density lipoproteins. GI VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
difficulties of the IPG technique that may be encountered not only with apoE, but also with other hydrophobic proteins 1321.
2.1 Materials Immobiline Dry Plates 4-7 and Ultrodex were from Pharmacia-LKB (Freiburg, Germany). Carrier ampholytes (Servalyt 4-7) and tris (hydroxymethyl) aminomethane (Tris) were from Serva (Heidelberg, Germany). Neuraminidase (Type VI from Clostridium perfringens) was provided by Sigma (Deisenhofen, Germany). Sodium decyl sulfate was from Eastman Kodak (Atlanta Chemie, Heidelberg). Polyvinylidene difluoride (PVDF) membranes (Immobilon) were from Millipore (Eschborn, Germany) and nylon membranes (Sartolon Plus) from Sartorius (Gottingen, Germany). Nitrocellulose filters, gelatin, and Tween 20 were from Bio-Rad Laboratories (Munich, Germany). Filter paper (2043bMgl) was from Schleicher and Schiill (Dassel, Germany). Goat anti-apoE was from Daiichi Pure Chemicals (WAK Chemie Medical, Bad Homburg, Germany). Biotinylated anti-goat IgG, avidin and biotinylated horseradish peroxidase (HRP) were from Vector Laboratories (Camon Laborservice, Wiesbaden, Germany). Urea (biochemical grade) and all other chemicals were obtained from E. Merck (Darmstadt, Germany).
2.2 Sample preparation 2.2.1 Delipidation Twenty pL plasma were added to 2 mL of a pre-chilled (-20" C) mixture of acetone:ethanol (1 :1 v/v), vortexed, and extracted twice at -20" C - first for 2 h, then for 1h - and finally with diethyl ether for 1 h (-20' C). The protein residue was evaporated under nitrogen, and, while still moist, dissolved in 100 pL of sample buffer (20 mM Tris-HCl, pH 10.0,6 M urea, 10 g/L sodium decyl sulfate, 10 mM dithiothreitol).
2.2.2 Neuraminidase treatment To 20 1 L plasma 80 pL of neuraminidase solution (3 U/mL in 50 mM sodium acetate, pH 5.5) were added, and the mixture was incubated overnight at 37" C. After delipidation, proteins were taken up in 100 pL sample buffer. 0173-0835/91/OlOl-0059 %3.50+.25/0
60
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( 5 mineach)inTTBS. Subsequently,itwasreactedfor 1 h with goat anti-apoE 1:SO0 v/v in TTBS with 10 g/L gelatin. After washing twice in TTBS, the membrane was incubated for 30 min with biotinylated rabbit anti-goat IgG (Vector Laboratories, 30 WLin 10 mL TTBS with 10 g/L gelatin), and again washed twiceinTTBS. Finally, the biotin label was traced with an avidin:biotinylated H R P complex (80 pL of each, avidin and biotinylated HRP, in 10 mLTTBS with lOg/Lgelatin)for 30 min. After washing in TTBS and rinsing in TBS, the membrane was stained for 15 to 30 min.
Immobiline Dry Plates, pH 4-7, were rehydrated in 8 M urea, 2 mMTris, 50 mMdithiothreito1 and0.2 % w/v Servalpt 4-7 for 4 h. Forty mg Ultrodex were suspended in 1 mL Servalyt 4-7 and 10 pL of this suspension were added to 30 pL of the delipidated samples, 20 pL of this mixture being applied onto the gel surface, about 10 mm from the cathode. Eight layers of filter paper (2043bMgl), soaked in bidistilled water, were used as electrode wicks. Focusing (15 "C) started with 400 V for 1 h, and continued at 5000 V for 4 h in a Desaphor HF electrophoresis chamber connected to a Desatronic 6000/100 power pack (Desaga, Heidelberg, Germany). 3 Results and discussion
2.4 Blotting
3.1 Patterns of apoE
Transfer onto PVDF was carried out by capillary blotting. The gel was equilibrated for 10 min in a solution of 120 mM acetic acid and 20 % v/v methanol, and the PVDF membrane, previously activated in methanol and soaked in phosphate buffer, was placed onto the gel surface. It was followed by a sandwich of two wet filter papers (2043bMgl), five dry filter papers, a 2 cm layer of paper towels, and a glass plate, weighted with approximately 7 g/cm2. Blotting proceeded for 2 h.
A typical immunoblot is shown in Fig. 1. The apoE isomorphs are recognized as distinct and intensely stained bands. Faint bands, cathodally to apoE4, have recurrently been observed. They are probably due to cross-reactivities of the commercially available primary antibody. Nevertheless, immunolocalization still affords unequivocal interpretation of apoE patterns. There has been no confusion due to apoA-I, proapoA-I or serum amyloid A which all focus close to apoE on conventional IEF of delipidated triglyceride-rich lipoproteins [26,271. Another weakly stained band is found between the positions of apoE3 and apoE4. Its intensity is increased in hypertriglyceridemic samples (cf. Fig. 1, third lane from left; and Fig. 3, second lane from left). This band has also been observed by others 126, 281 and is apparently due to partial proteolytic cleavage of apoE.
2.5 Immunoassay for apoE 2.5.1 Solutions The TBS buffer consisted of 20 mM Tris-HC1, pH 7.5,0.5 M NaCl and 0.1 g/L Thimerosal. In the TTBS buffer, TBS additionally contained 0.05 % v/v Tween 20. Two solutions were prepared for staining: (i) 120 mg 4-chloro- I-naphthol were dissolved in 40 mL ice-cold methanol and (ii) immediately prior to use 120 pL H 2 0 2 (30 %, w/w) were added to 200 mL TBS and mixed with solution (i).
2.5.2 Procedure The membrane was quenched for 30 min in TBS supplemented with 10 g/L polyvinylpyrrolidone and washed twice
In plasma, negative charges are conferred on the allelic forms of apoE by avarying number of sialic acid residues [33]. This has confused phenotyping in many CA methods because the monosialo forms of apoE3 and apoE4 cofocus with the asialo forms apoE3 and apoE2, respectively. In IPG the positions of the monosialo isoforms are slightly anodic to the proximate major asialo isoforms. Thus, it is possible to distinguish sialylated apoE3 from asialo apoE2 (cf.Fig. 1, third lane from left; Fig. 3, second lane from left) as well as sialylated apoE4 from asialo apoE3 (cf.Fig. 2, third lane from left). However,it appears that the resolution attained with delipidated VLDL
Figure I . Immunoblotting of apoE. Plasma was delipidated with organic solvents. The proteins were redissolved in denaturing sample buffer, focused in a composite CA-IPG system, pH 4-7, and blotted to a PVDF membrane. Immunostaining was performed with an avidin biotin complex. For further methodical details see Section 2.5.2.
Electrophoresis 1991, 12.59-63
Immunoblotting of apolipoprotein E
61
Figure 2. Pretreatment of samples with neuraminidase. Plasma samples were digested with neuraminidase, delipidated, and analyzed for apoE isoforms by immunoblotting: + neuraminidase digested samples, - controls without neuraminidase.
Figure 3. Immunoblotting of apoE. Experimental conditions as described in Section 2. Transfer membrane was nylon instead of PVDF.
[30] is partially lost in this system, most likely because ofdiffusion during the blotting step. In cases where uncertainty due to the presence of sialic acid residues remains, the intensity ofthe sialylated isomorphs can be reduced by digesting the samples with neuraminidase prior to delipidation (cJ:Fig. 2). Yet, since normally there is little doubt about the precise phenotype, incubation with neuraminidase is not often required.
3.2 Optimization During the development of this method we have identified a number of crucial experimental parameters. It has been emphasized that, in IPG, hydrophobic proteins may entail serious complications because they scarcely penetrate the gel matrix 1321. This has also been experienced in our work with apoE. The most common remedy to this problem, including CA into the gel andinto the sample buffer [321,did not help until we turned to the delipidation of the samples.
Since the method could be expedited considerably by skipping over the delipidation step, several ways of pretreating samples with detergents have been evaluated. Towards this end, the sample buffer (Tris, urea, and dithiothreitol) was supplemented with the following additives: sodium decyl sulfate (13 g/L), sodium dodecyl sulfate (13 g/L), Triton X-100 (1.3 %, v/v), Triton X-114 (1.3 %, v/v), Genapol X-080 (0.3 %, v/v), Brij 35 (0.5 %, v/v), Tween 20 (0.5 %, v/v), Tween 80 (0.5 %, v/v), Nonidet P-40 (0.5 %, v/v), octyl-P-Dglucopyranoside (40 g/L), and Tergitol 15-S-9(1 % v/v). In addition, denaturation with guanidine-HC1, as proposed by Havekes et al. [281, was tested. Without prior organic solvent extraction none of these pretreatments effected entry of apoE into the IPG matrices, irrespective of whether we loaded high or low amounts of protein to the gels. Inclusion of nonionic detergents in the rehydration solution as well as in the sample buffer improved the entry of apoE into the gels even with undelipidated samples. However, the patterns obtained under
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W. Marz et al.
these conditions may become difficult to interpret.'Hence, it appears that currently there is no reproducible way to bypass delipidation if apoE phenotypes are to be analyzed in IPGs. This is in line with a previous report on phenotyping of apoE from VLDL in IPGs [301, and most authors working with CA methods for apoE immunoblotting consider delipidation or denaturation absolutely necessary 126-281. However, Kamboh et al. 1291 recently claimed that dialysis of the sample against phosphate buffer replaced delipidation.
For the solubilization of the delipidated plasma proteins we have compared different buffer formulations with respect to pH values, ionic strengths and additives (urea, sodium dodecyl sulfate, sodium decyl sulfate and guanidine-HC1). Far the best results have been obtained with a slight modification (20 instead of 100 mM Tris) of a recipe by Menzel and Utermann [261. Addition of Ultrodex and of CA to the samples significantly lessens protein precipitation at the beginning of IEF [32, 341. In our hands, direct application of the sample onto the gel surface was preferred to application onto sample applicator strips. The composition of the IPG gel is of major importance. As with other hydrophobic proteins, admixture of CA to IPG is essential for penetration of apoE into the matrix [30]. Dithiothreitol was included as a reducing agent because disulfide stabilized complexes involving apoE have been reported 135, 361, and because oxidation of cysteine residues is known to cause the formation of protein oligomers and artifactual band splitting in IPGs [371. Among numerous blotting membranes, nitrocellulose, nylon, and PVDF have been compared. Figure 3 shows a blot to a nylon membrane that has been obtained under otherwise identical conditions. Although phenotyping is still reliable, the signal-to-background ratio is lower than on PVDF.
Electrophoresis 1991,12,59-63
al. [30]. Running delipidated VLDL in IPGs, these authors unequivocally detected apoE4 in Coomassie Blue stained gels. Thus, at the moment, the afflictionsintroduced by polyol compounds remain unresolved. It appears that the sensitivity to liquid exudation differs between different lots of Immobiline gels. Therefore, careful pretesting may be advantageous. As has been pointed out, the PVDF membrane is most sensitive to disturbances from liquid exudation. However, since it warrants sensitive and reproducible visualization of apoE, focusing should be terminated early, for instance after 3 ?hh, in experiments where excessive liquid exudation occurs.
3.4 Visualization To visualize apoE, we have preferred a sensitive avidin-biotin complex because with conventional two-step peroxidase staining, bands were sometimes too faint to allow reliable phenotyping. This does not rule out that satisfactory results may be obtained with many other immunoassays. It should be born in mind that the ~4 allele is associated with low apoE levels; thus, in E3/4 heterozygotes the apoE4 band is less intense than the apoE3 band, and apoE4 may easily be overlooked if immunoprobing is not sensitive enough. Recently, apoE genotyping using allele-specific oligonucleotide hybridization [41, 421 or cleavage with the restriction enzyme HhaI [431 of in vitro amplified DNA has been reported. These methods are of great potential value in the rapid discrimination of apoE variants. However, if genotyping alone is performed with probes or endonucleases specific for the common polymorphic sites (residues 112 and 158) some rare charge variants may not be detected or will be misclassified. Therefore, conventional phenotyping at the protein level and genotyping are complementary rather than exclusive approaches.
3.3 Liquid exudation Another major problem with hybrid IPG-CA gels has been the exudation of liquid from the gel surface [381, which occurs after approximately 3% h of focusing at 5000 V. Intriguingly, although drops are completely washed away with acetic acid/ methanol, we obtained poor transfer ofprotein to PVDF in the regions of the liquid drops. Since this phenomenon did not arise with nitrocellulose or nylon membranes it seems unlikely that proteins are simply lost with the spilled liquid. In line with observations by other investigators 138, 391, the addition of glycerol (30 %, vfv) efficiently suppressed liquid exudation and instantaneously restored protein transfer to PVDF [401. However, the addition of glycerol, as well as of sucrose or sorbitol, resulted in the complete disappearance of the apoE4 isoform and thus precluded precise phenotyping. Moreover, in the presence of glycerol the position of apoE bands shifted towards the cathode. Since the runs with glycerol had been carried out at 50 000 Vh (instead of 20 000 Vh in the standard procedure) and sharp zones were obtained, we do not believe that we failed to reach equilibrium. Therefore, other mechanisms such as structural changes and effects on the equilibrium of the reactions determining apparent isoelectric points may be responsible for the observed shifts. The disappearance of the E4 band in the presence of glycerol is in conflict with the results obtained by Baumstark et
4 Concluding remarks The described immunoblotting method affords apoE phenotyping without isolation of triglyceride-rich lipoproteins. Commercially available gels can be used and, therefore, the method is suitable for clinical application. Further systematic studies will have to address the problems due to liquid exudation and the effects of high concentrations of polyols. Moreover, circumventing delipidation by including appropriate detergents into the sample buffer as well as into the rehydration solution should be attempted. At present, however, we are convinced that the degree of standardization that has been attained in our procedure will warrant reliable performances in any laboratory. The authors thank Dr. Manfred W.Baumstark, University of Freiburg, f o r useful discussions. Received July 3 , 1990
5 References 111 Rall,S.C.,Weisgraber,K. H. andMahley,R. W.,J.Biol. Chem. 1982, 257,4171-4178.
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[21 Paik, Y. K., Chang, D. J., Reardon, C. A., Davies, G. E., Mahley, R. W. and Taylor, J. M., Proc. Nutl. Acad. Sci. USA 1985, 82, 3445-3449. [31 Mahley, R. W., Science 1988,240, 622-630. [41 Utermann, G., Am. Heart J. 1987,113.433-440. [5] Gregg, R. E. and Brewer, H. B., Clin. Chem. 1988,34, B28-B32. [61 Davignon, J., Gregg, R. E. and Sing, C. F., Arteriosclerosis 1988,8, 1-21. 1981, 171 Weisgraber,K.H.,Rall,S.C.andMahley,R.W.,J.Biol.Chem. 256,9077-9083. 181 Rall, S. C., Weisgraber, K. H., Innerarity, T. L., Bersot, T. P. and Mahley, R. W., J. Clin. Invest. 1983, 72, 1288-1297. I91 Weisgraber, K. H., Rall, S. C., Innerarity, T. L., Mahley, R. W., Kuusi, T. and Ehnholm, C., J. Clin. Invest. 1984, 73, 1024-1033. 1101 Yamamura, T., Yamamato, A., Hiramori, K. and Nambu, S., Atherosclerosis 1984,50, 159-172. [ I 11 Ghiselli, G. V., Gregg, R. E. and Brewer, H. B., Biochim. Biophys. ActU 1984,794,333-339. [ 121 Wardell, M. R., Weisgraber, K. H., Havekes, L. M. and Rall, S. C., J. Biol. Chem. 1989.264.21205-21210. I 1 31 Wardell, M. R., Brennan, S. O., Janus, E. D., Fraser, R. and Carrel], R. W., J . Clin. Invest. 1987,80, 483-490. [141 Tajima, S., Yarnamura, T., Menju, M. and Yamamoto, A., J. Biochem. 1989,105,249-253. [ 151 Schneider, W. J., Kovanen, P. T.,Brown,M. S., Goldstein, J. L.,Utermann, G., Weber, W., Havel, R. J., Kotite, L., Kane,J. P., Innerarity, T. L. and Mahley, R. W., J. Clin Invest. 1981,68, 1075-1085. [ 161 Lalzar, A., Weisgraber, K. H., Rall, S. C., Giladi,H., Innerarity,T. L., Levanon, A. Z., Boyles, J. K., Amit, B., Gorecki, M., Mahley, R. W. and Vogel, T., J. B i d . Chem. 1988,263,3542-3545. [ I71 Weisgraber, K. H.,Innerarity, T. L. andMahley, R. W.,J. Biol. Chem. 1982,257,2518-2521. [IS] Utermann, G., Hees, M. and Steinmetz, A., Nature 1977 269, 604-607. 1191 Boerwinkle, E. and Utermann, G., Am. J . Hum. Genet. 1988, 42, 104- 112. [201 Utermann, G., Albrecht, G. and Steinmetz, A,, Clin.Gen. 1978,14, 35 1-358. [211 Warnick, G. R.,Mayfield, C., Albers, J. J. and Hazzard, W. R., Clin. Chem. 1979,25,279-284. [221 Utermann, G., Steinmetz, A. and Weber, W., Hum. Genet. 1982,60, 344-35 1.
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[231 Bouthillier, D., Sing, C. F. and Davignon, J., J . Lipid Res. 1983,24, 1060-1069. 1241 Eto, M., Watanabe, K. and Ishii, K., Clin. Chim. A d a 1985, 149, 21-28. 1251 Kane, J. W. and Gowland, E., Ann. Clin. Biochem. 1986, 23, 509-513. 1261 Menze1,H:J. andutermann, G., Elecrrophoresis 1986,7,492-495. [271 Steinmetz, A.,J. LipidRes. 1987,28, 1364-1370. [281 Havekes, L. M., de Knijff, P., Beisiegel, U., Havinga, J., Smit, M. and Klasen, E., J. Lipid Res. 1987,28,455-463. [291 Kamboh, M. I., Ferrell, R. E. and Kottke, B. J. Lipid Res. 1988,29, 1535-1543. I301 Baumstark, M. W., Berg, A., Halle, M. and Keul, J., Electrophoresis 1988,9,576-579. 1311 Marz, W., Cezanne, S. and GroB, W., in: Radola, B. J. (Ed.), Electrophoresis Forum ’89, Technical University Munich 1989, pp. 403-407. [321 Righetti, P. G., Chiari, M. and Gelfi, C., Electrophoresis 1988, 9, 65-73. [331 Zannis, J. L. and Breslow,J. L., Biochemistry 198 1,20,1033- 1041. 1341 Gorg, A,, Postel, W. and Giinther, S., Electrophoresis 1988, 9, 53 1-546. [35] Weisgraber, K. H. and Mahley, R. W., J. Bi d . Chem. 1978, 253, 6281-6288. [361 Tada, N., Fidge, N. and Nestel, P., Biochem. Biophys. Res. Commun. 1979,90,291-304. [371 Altland, K., Becker, P., Rossmann, U. and Bjellqvist B., Electrophoresis 1988,9,474-485. [381 Altland, K., Hackler, R. and Rossmann, U., Electrophoresis 1986,7, 25 1-259. 1391 Patestos N. P., Zok, R., and Radola, B. J. in: Radola, B. J. (Ed.), Electrophoresis Forum ’89, Technical University Munich 1989, pp. 368-370. 1401 Kinzkofer-Peresch, A., Patestos, N. P., Fauth,M., Kogel, F., Zok, R. and Radola, B. J., Electrophoresis 1988,9,497-511. [4 11 Smeets, H. J. M., Poddighe, J., Stuyt, P. M. J., Stalenhoef, A. F. H., Ropers,H.H. and Wieringa,B., J.LipidRes. 1988,29,123 1-1237. [42] Weisgraber, K. H., Newhouse, Y. M. and Mahley, R. W., Biochem. Biophys. Res. Commun. 1988,157, 1212-1217. [43] Hixson, J.-E. and Vernier, D. T., J. Lipid Res. 1990,31,545-548.
Immunoblotting of apolipoprotein E
59
Winfried Marz Sabine Cezanne Werner GroB
Phenotyping of apolipoprotein E by immunoblotting in immobilized pH gradients
Gustav Embden-Centerof Biological Chemistry, J. W. Goethe-University, Frankfurt/Main
An immunoblotting method for the determination of apolipoprotein E (apoE) phenotypes has been developed. Delipidated plasma proteins are focused in an immobilized pH gradient, and transferred to polyvinylidene difluoride (PVDF) membranes. ApoE isomorphs are identified by immunoperoxidase staining. The method allows reproducible assignment of apoE phenotypes without isolation of triglyceride-rich lipoproteins. Only small amounts of serum are required. There are several important steps in the procedure: (i) delipidation is indispensable, (ii) carrier ampholytes have to be added to the gels and to the sample buffer, and, (iii) on immunostaining, polyvinylidene difluoride membranes provide an excellent signal-tobackground ratio.
1 Introduction Apolipoprotein E (apoE) is a well-characterized 34 kDa glycoprotein, associated with triglyceride-rich and high density lipoproteins [ 1-31. It is a ligand for low density lipoprotein (LDL) receptors and hepatic lipoprotein remnant receptors, and plays a key role in the catabolism of triglyceride-rich lipoproteins 14-61. Three alleles ( ~ 2~, 3~, 4at) the apoE locus give rise to three homozygous (E2/2, E3/3, and E4/4) and three heterozygous phenotypes (E3/2, E4/2, and E4/3). This polymorphism is due to cysteine-arginine interchanges at amino acid residues 1 12 and 158. ApoE4 contains arginine and E2 contains cysteine at both sites, whereas E3 has cysteine at position 112 and arginine at position 158 [71. Other rare alleles have also been reported [8- 141.ApoE2 is defective in binding to apoB, E receptors 115-171. Because the clearance of both chylomicron and very low density lipoprotein (VLDL) remnants depends on functional apoE, homozygotes for apoE2 accumulate remnant particles in their circulation. Concomitantly, the flux of dietary cholesterol into the liver is decreased and hepatic apoB,E receptors are up-regulated. Therefore, E2 homozygotes have low LDL cholesterol and apoB levels. However, in response to some additional genetic, metabolic or environmental factors, a small fraction of E2 homozygotes expresses type I11 hyperlipoproteinemia [4-6, 181. The ~4 allele exerts an opposite effect on lipoprotein levels: It is associated with high LDL cholesterol and apo B levels, and appears to predispose to premature atherosclerosis [4-6, 191. Conventionally, apoE phenotyping has been performed by isoelectric focusing (IEF) of delipidated triglyceride-rich lipoproteins 120-251. Immunoblotting of apoE after IEF in carrier ampholyte (CA)-based pH gradients has been reported [26-291. Moreover, it has been demonstrated that IEF of delipidated VLDL in immobilized pH gradients (IPG) affords improved apoE phenotyping [301. We have developed a method that combines the advantages of IEF in IPGs with thoseofimmunoblotting [3 I]. Here we report on the optimization of this procedure. Particular emphasis is placed on the methodical Correspondence: Prof. Dr. W. Grofi, Gustav Embden-Center of Biological Chemistry, Johann Wolfgang Goethe-University, Theodor Stern-Kai 7, D-6000 Frankfurt am Main, Germany Abbreviations: apo, apolipoprotein; CA, carrier ampholytes; HRP, horseradish peroxidase; IEF, isoelectric focusing; IPG, immobilized pH gradient; PVDF, polyvinylidene difluoride; Tris, tris (hydroxymethyl) aminomethane; VLDL, LDL, HDL, very low, low, and high density lipoproteins. GI VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
difficulties of the IPG technique that may be encountered not only with apoE, but also with other hydrophobic proteins 1321.
2.1 Materials Immobiline Dry Plates 4-7 and Ultrodex were from Pharmacia-LKB (Freiburg, Germany). Carrier ampholytes (Servalyt 4-7) and tris (hydroxymethyl) aminomethane (Tris) were from Serva (Heidelberg, Germany). Neuraminidase (Type VI from Clostridium perfringens) was provided by Sigma (Deisenhofen, Germany). Sodium decyl sulfate was from Eastman Kodak (Atlanta Chemie, Heidelberg). Polyvinylidene difluoride (PVDF) membranes (Immobilon) were from Millipore (Eschborn, Germany) and nylon membranes (Sartolon Plus) from Sartorius (Gottingen, Germany). Nitrocellulose filters, gelatin, and Tween 20 were from Bio-Rad Laboratories (Munich, Germany). Filter paper (2043bMgl) was from Schleicher and Schiill (Dassel, Germany). Goat anti-apoE was from Daiichi Pure Chemicals (WAK Chemie Medical, Bad Homburg, Germany). Biotinylated anti-goat IgG, avidin and biotinylated horseradish peroxidase (HRP) were from Vector Laboratories (Camon Laborservice, Wiesbaden, Germany). Urea (biochemical grade) and all other chemicals were obtained from E. Merck (Darmstadt, Germany).
2.2 Sample preparation 2.2.1 Delipidation Twenty pL plasma were added to 2 mL of a pre-chilled (-20" C) mixture of acetone:ethanol (1 :1 v/v), vortexed, and extracted twice at -20" C - first for 2 h, then for 1h - and finally with diethyl ether for 1 h (-20' C). The protein residue was evaporated under nitrogen, and, while still moist, dissolved in 100 pL of sample buffer (20 mM Tris-HCl, pH 10.0,6 M urea, 10 g/L sodium decyl sulfate, 10 mM dithiothreitol).
2.2.2 Neuraminidase treatment To 20 1 L plasma 80 pL of neuraminidase solution (3 U/mL in 50 mM sodium acetate, pH 5.5) were added, and the mixture was incubated overnight at 37" C. After delipidation, proteins were taken up in 100 pL sample buffer. 0173-0835/91/OlOl-0059 %3.50+.25/0
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2.3 IEF
( 5 mineach)inTTBS. Subsequently,itwasreactedfor 1 h with goat anti-apoE 1:SO0 v/v in TTBS with 10 g/L gelatin. After washing twice in TTBS, the membrane was incubated for 30 min with biotinylated rabbit anti-goat IgG (Vector Laboratories, 30 WLin 10 mL TTBS with 10 g/L gelatin), and again washed twiceinTTBS. Finally, the biotin label was traced with an avidin:biotinylated H R P complex (80 pL of each, avidin and biotinylated HRP, in 10 mLTTBS with lOg/Lgelatin)for 30 min. After washing in TTBS and rinsing in TBS, the membrane was stained for 15 to 30 min.
Immobiline Dry Plates, pH 4-7, were rehydrated in 8 M urea, 2 mMTris, 50 mMdithiothreito1 and0.2 % w/v Servalpt 4-7 for 4 h. Forty mg Ultrodex were suspended in 1 mL Servalyt 4-7 and 10 pL of this suspension were added to 30 pL of the delipidated samples, 20 pL of this mixture being applied onto the gel surface, about 10 mm from the cathode. Eight layers of filter paper (2043bMgl), soaked in bidistilled water, were used as electrode wicks. Focusing (15 "C) started with 400 V for 1 h, and continued at 5000 V for 4 h in a Desaphor HF electrophoresis chamber connected to a Desatronic 6000/100 power pack (Desaga, Heidelberg, Germany). 3 Results and discussion
2.4 Blotting
3.1 Patterns of apoE
Transfer onto PVDF was carried out by capillary blotting. The gel was equilibrated for 10 min in a solution of 120 mM acetic acid and 20 % v/v methanol, and the PVDF membrane, previously activated in methanol and soaked in phosphate buffer, was placed onto the gel surface. It was followed by a sandwich of two wet filter papers (2043bMgl), five dry filter papers, a 2 cm layer of paper towels, and a glass plate, weighted with approximately 7 g/cm2. Blotting proceeded for 2 h.
A typical immunoblot is shown in Fig. 1. The apoE isomorphs are recognized as distinct and intensely stained bands. Faint bands, cathodally to apoE4, have recurrently been observed. They are probably due to cross-reactivities of the commercially available primary antibody. Nevertheless, immunolocalization still affords unequivocal interpretation of apoE patterns. There has been no confusion due to apoA-I, proapoA-I or serum amyloid A which all focus close to apoE on conventional IEF of delipidated triglyceride-rich lipoproteins [26,271. Another weakly stained band is found between the positions of apoE3 and apoE4. Its intensity is increased in hypertriglyceridemic samples (cf. Fig. 1, third lane from left; and Fig. 3, second lane from left). This band has also been observed by others 126, 281 and is apparently due to partial proteolytic cleavage of apoE.
2.5 Immunoassay for apoE 2.5.1 Solutions The TBS buffer consisted of 20 mM Tris-HC1, pH 7.5,0.5 M NaCl and 0.1 g/L Thimerosal. In the TTBS buffer, TBS additionally contained 0.05 % v/v Tween 20. Two solutions were prepared for staining: (i) 120 mg 4-chloro- I-naphthol were dissolved in 40 mL ice-cold methanol and (ii) immediately prior to use 120 pL H 2 0 2 (30 %, w/w) were added to 200 mL TBS and mixed with solution (i).
2.5.2 Procedure The membrane was quenched for 30 min in TBS supplemented with 10 g/L polyvinylpyrrolidone and washed twice
In plasma, negative charges are conferred on the allelic forms of apoE by avarying number of sialic acid residues [33]. This has confused phenotyping in many CA methods because the monosialo forms of apoE3 and apoE4 cofocus with the asialo forms apoE3 and apoE2, respectively. In IPG the positions of the monosialo isoforms are slightly anodic to the proximate major asialo isoforms. Thus, it is possible to distinguish sialylated apoE3 from asialo apoE2 (cf.Fig. 1, third lane from left; Fig. 3, second lane from left) as well as sialylated apoE4 from asialo apoE3 (cf.Fig. 2, third lane from left). However,it appears that the resolution attained with delipidated VLDL
Figure I . Immunoblotting of apoE. Plasma was delipidated with organic solvents. The proteins were redissolved in denaturing sample buffer, focused in a composite CA-IPG system, pH 4-7, and blotted to a PVDF membrane. Immunostaining was performed with an avidin biotin complex. For further methodical details see Section 2.5.2.
Electrophoresis 1991, 12.59-63
Immunoblotting of apolipoprotein E
61
Figure 2. Pretreatment of samples with neuraminidase. Plasma samples were digested with neuraminidase, delipidated, and analyzed for apoE isoforms by immunoblotting: + neuraminidase digested samples, - controls without neuraminidase.
Figure 3. Immunoblotting of apoE. Experimental conditions as described in Section 2. Transfer membrane was nylon instead of PVDF.
[30] is partially lost in this system, most likely because ofdiffusion during the blotting step. In cases where uncertainty due to the presence of sialic acid residues remains, the intensity ofthe sialylated isomorphs can be reduced by digesting the samples with neuraminidase prior to delipidation (cJ:Fig. 2). Yet, since normally there is little doubt about the precise phenotype, incubation with neuraminidase is not often required.
3.2 Optimization During the development of this method we have identified a number of crucial experimental parameters. It has been emphasized that, in IPG, hydrophobic proteins may entail serious complications because they scarcely penetrate the gel matrix 1321. This has also been experienced in our work with apoE. The most common remedy to this problem, including CA into the gel andinto the sample buffer [321,did not help until we turned to the delipidation of the samples.
Since the method could be expedited considerably by skipping over the delipidation step, several ways of pretreating samples with detergents have been evaluated. Towards this end, the sample buffer (Tris, urea, and dithiothreitol) was supplemented with the following additives: sodium decyl sulfate (13 g/L), sodium dodecyl sulfate (13 g/L), Triton X-100 (1.3 %, v/v), Triton X-114 (1.3 %, v/v), Genapol X-080 (0.3 %, v/v), Brij 35 (0.5 %, v/v), Tween 20 (0.5 %, v/v), Tween 80 (0.5 %, v/v), Nonidet P-40 (0.5 %, v/v), octyl-P-Dglucopyranoside (40 g/L), and Tergitol 15-S-9(1 % v/v). In addition, denaturation with guanidine-HC1, as proposed by Havekes et al. [281, was tested. Without prior organic solvent extraction none of these pretreatments effected entry of apoE into the IPG matrices, irrespective of whether we loaded high or low amounts of protein to the gels. Inclusion of nonionic detergents in the rehydration solution as well as in the sample buffer improved the entry of apoE into the gels even with undelipidated samples. However, the patterns obtained under
62
W. Marz et al.
these conditions may become difficult to interpret.'Hence, it appears that currently there is no reproducible way to bypass delipidation if apoE phenotypes are to be analyzed in IPGs. This is in line with a previous report on phenotyping of apoE from VLDL in IPGs [301, and most authors working with CA methods for apoE immunoblotting consider delipidation or denaturation absolutely necessary 126-281. However, Kamboh et al. 1291 recently claimed that dialysis of the sample against phosphate buffer replaced delipidation.
For the solubilization of the delipidated plasma proteins we have compared different buffer formulations with respect to pH values, ionic strengths and additives (urea, sodium dodecyl sulfate, sodium decyl sulfate and guanidine-HC1). Far the best results have been obtained with a slight modification (20 instead of 100 mM Tris) of a recipe by Menzel and Utermann [261. Addition of Ultrodex and of CA to the samples significantly lessens protein precipitation at the beginning of IEF [32, 341. In our hands, direct application of the sample onto the gel surface was preferred to application onto sample applicator strips. The composition of the IPG gel is of major importance. As with other hydrophobic proteins, admixture of CA to IPG is essential for penetration of apoE into the matrix [30]. Dithiothreitol was included as a reducing agent because disulfide stabilized complexes involving apoE have been reported 135, 361, and because oxidation of cysteine residues is known to cause the formation of protein oligomers and artifactual band splitting in IPGs [371. Among numerous blotting membranes, nitrocellulose, nylon, and PVDF have been compared. Figure 3 shows a blot to a nylon membrane that has been obtained under otherwise identical conditions. Although phenotyping is still reliable, the signal-to-background ratio is lower than on PVDF.
Electrophoresis 1991,12,59-63
al. [30]. Running delipidated VLDL in IPGs, these authors unequivocally detected apoE4 in Coomassie Blue stained gels. Thus, at the moment, the afflictionsintroduced by polyol compounds remain unresolved. It appears that the sensitivity to liquid exudation differs between different lots of Immobiline gels. Therefore, careful pretesting may be advantageous. As has been pointed out, the PVDF membrane is most sensitive to disturbances from liquid exudation. However, since it warrants sensitive and reproducible visualization of apoE, focusing should be terminated early, for instance after 3 ?hh, in experiments where excessive liquid exudation occurs.
3.4 Visualization To visualize apoE, we have preferred a sensitive avidin-biotin complex because with conventional two-step peroxidase staining, bands were sometimes too faint to allow reliable phenotyping. This does not rule out that satisfactory results may be obtained with many other immunoassays. It should be born in mind that the ~4 allele is associated with low apoE levels; thus, in E3/4 heterozygotes the apoE4 band is less intense than the apoE3 band, and apoE4 may easily be overlooked if immunoprobing is not sensitive enough. Recently, apoE genotyping using allele-specific oligonucleotide hybridization [41, 421 or cleavage with the restriction enzyme HhaI [431 of in vitro amplified DNA has been reported. These methods are of great potential value in the rapid discrimination of apoE variants. However, if genotyping alone is performed with probes or endonucleases specific for the common polymorphic sites (residues 112 and 158) some rare charge variants may not be detected or will be misclassified. Therefore, conventional phenotyping at the protein level and genotyping are complementary rather than exclusive approaches.
3.3 Liquid exudation Another major problem with hybrid IPG-CA gels has been the exudation of liquid from the gel surface [381, which occurs after approximately 3% h of focusing at 5000 V. Intriguingly, although drops are completely washed away with acetic acid/ methanol, we obtained poor transfer ofprotein to PVDF in the regions of the liquid drops. Since this phenomenon did not arise with nitrocellulose or nylon membranes it seems unlikely that proteins are simply lost with the spilled liquid. In line with observations by other investigators 138, 391, the addition of glycerol (30 %, vfv) efficiently suppressed liquid exudation and instantaneously restored protein transfer to PVDF [401. However, the addition of glycerol, as well as of sucrose or sorbitol, resulted in the complete disappearance of the apoE4 isoform and thus precluded precise phenotyping. Moreover, in the presence of glycerol the position of apoE bands shifted towards the cathode. Since the runs with glycerol had been carried out at 50 000 Vh (instead of 20 000 Vh in the standard procedure) and sharp zones were obtained, we do not believe that we failed to reach equilibrium. Therefore, other mechanisms such as structural changes and effects on the equilibrium of the reactions determining apparent isoelectric points may be responsible for the observed shifts. The disappearance of the E4 band in the presence of glycerol is in conflict with the results obtained by Baumstark et
4 Concluding remarks The described immunoblotting method affords apoE phenotyping without isolation of triglyceride-rich lipoproteins. Commercially available gels can be used and, therefore, the method is suitable for clinical application. Further systematic studies will have to address the problems due to liquid exudation and the effects of high concentrations of polyols. Moreover, circumventing delipidation by including appropriate detergents into the sample buffer as well as into the rehydration solution should be attempted. At present, however, we are convinced that the degree of standardization that has been attained in our procedure will warrant reliable performances in any laboratory. The authors thank Dr. Manfred W.Baumstark, University of Freiburg, f o r useful discussions. Received July 3 , 1990
5 References 111 Rall,S.C.,Weisgraber,K. H. andMahley,R. W.,J.Biol. Chem. 1982, 257,4171-4178.
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