[20]
IMMUNOISOLATION OF P 4 5 0 AUTOANTIGENS
201
mainly recognize native cytochromes P450,7 the antibodies directed against P450 fusion proteins recognize mainly denatured cytochromes P450. We were also successful in generating antibodies against P450 fusion proteins containing almost full-length P450IIC6 or P450IIC7 fusion protein. The antibodies recognized these proteins in microsomes. Thus, fusion proteins can be used to generate antibodies against a cytochrome P450 which is not available in a purified form in the laboratory. Acknowledgments We thank Mrs. I. Bfhm and Mrs. H. Steedfor typingthe manuscript.This work was supported by the DeutscheForschungsgemeinschaft(SFB 302).
[20] I m m u n o i s o l a t i o n of H u m a n M i c r o s o m a l C y t o c h r o m e s P 4 5 0 Using A u t o a n t i b o d i e s
By
ULRICH M.
ZANGER
Introduction Human cytochrome P450 isozymes of three P450 subfamilies have recently been shown to be specifically recognized by certain types of circulating autoantibodies, which occur in patients with either drug-induced or idiopathic chronic active forms of hepatitis (Table I; see also [21], this volume). All these P450 isozymes are mainly expressed in the liver, they metabolize xenobiotic compounds, and their catalytic function is potently inhibited by the respective autoantibodies. Besides the interest evoked by these autoantibodies in the mechanisms leading to autoimmunity and drug-induced hepatitis, they have proved to be highly valuable immunological tools of amazing specificity and potency. The protocols presented in this chapter describe the use of autoantisera to immunoisolate P450 proteins from solubilized microsomes. They may of course be applicable to other types of autoantibodies as well as to antibodies raised in rabbits. A microscale procedure is presented, which allows one to detect and semiquantitatively isolate microsomal antigens. By immunopurification on a larger scale, sufficient amounts of highly pure antigen can be obtained for further protein chemical analysis and for the production of antibodies. These methods have been used with the socalled anti-LKM (anti-liver/kidney microsome) antibodies type 1 and 2 to detect and identify their autoantigens, the liver microsomal P450IID6 and METHODS IN ENZYMOLOGY, VOL. 206
Copyright© 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.
202
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[20]
TABLE I HUMAN P450 ISOZYMESRECOGNIZED BY AUTOANTIBODIES Autoantibody Anti-LKMI°
Anti-LKM2a
Anti-LMa Idiopathic chronic active (4) or druginduced (5) (dihydralazine) hepatitis P450IA2 (4), P450PA (5) IA2 Ethoxyresorufin (4,5), phenacetin
Liver disease
Autoimmune chronic active hepatitis type I~ (1,2)
Drug-induced (tienilic acid) hepatitis (3)
Recognized human P450 b P450 classifiedc Substrates
P450dbl (6-8), P450bufI (9) IID6 Debrisoquine, sparteine, bufuralol (12)
P450-8 (10), P450meph (11) IIC8/9/10 e Tienilic acid (10), mephenytoin (11)
Comments
Genetically polymorphicd
Genetically polymorphic e
(5) Inducible (4,13)
a Abbreviations are based on immunohistochemical classification: anti-LKMl~, anti-liver/ kidney microsome antibody type 1 (1,2) or 2 (3); anti-LM, antiliver microsome antibody (4,5). See Ref. (14) for a recent review. b Names of human P450 isozymes used by authors describing recognition by autoantibodies. c Classification according to the most recent proposal for cytochrome P450 nomenclature (15) P450IID6 has previously been designated as P450IID1 (12). d P450IID6 is not expressed in 5-10% of Caucasians [debrisoquine/sparteine type genetic polymorphism of drug oxidation; see Meyer et al. (12) for review]. e Anti-LKM2 possibly recognizes several closely related P450 isozymes of the same subfamily which are presently referred to as P450IIC8/9 (12) or P450IIC8D/10 (5). One of these or a highly similar isozyme appears to be affected by the mephenytoin type genetic polymorphism (11,12,16). f Key to references: (1) M. Rizzetto, G. Swana, and D. Doniach, Clin. Exp. lmmunol. 15, 331 (1973); (2) J. C. Homberg, N. Abuaf, O. Bernard, S. Islam, F. Alvarez, S. H. Khalil, R. Poupon, F. Darnis, V.-G. Levy, P. Grippon, P. Opolon, J. Beruuau, J.-P. Benhamou, and D. Alagille, Hepatology 7, 1333 (1987); (3) J.-C. Homberg, C. Andre, and N. Abuaf, Clin. Exp. Immunol. 55, 561 (1984); (4) M. P. Manns, K. J. Griffin, L. C. Quattrochi, M. Sacher, H. Thaler, R. H. Tukey, and E. F. Johnson, Arch. Biochem. Biophys. 7,80, 229 (1990). (5) M. Bourdi, D. Larrey, J. Nataf, J. Bernuau, D. Pessayre, M. Iwasalli, F. P. Guengerich, and P. H. Beaune, J. Clin. Invest. 85, 1967 (1990); (6) U. M. Zanger, H. P. Hauri, J. Loeper, J. C. Homberg, and U. A. Meyer, Proc. Natl. Acad. Sci. U.S.A. 85, 8256 (1988); (7) M. Manns, E. F. Johnson, K. J. Griffin, E. M. Tan, and K. F. Sullivan, J. Clin. Invest. 83, 1066 (1989); (8) M. Gueguen, A. M. Yamamoto, O. Benard, and F. Alvarez, Biochem. Biophys. Res. Commun. 159, 542 (1989); (9) L. Kiffel, J. Loeper, J. C. Homberg, and J. P. Leroux, Biochem. Biophys. Res. Commun. 159, 283 (1989); (10) P. Beaune, P. M. Dansette, D. Mansuy, L. Kiffel, M. Finck, C. Amar, J. P. Leroux, and J. C. Homberg, Proc. Natl. Acad. Sci. U.S.A. 84, 551 (1987); (11) U. T. Meier and U. A. Meyer, Biochemistry 26, 8466 (1987); (12)
[20]
IMMUNOISOLATION OF P450 AUTOANTIGENS
203
P450meph, respectively (Table I). Anti-LKM antibodies provided unique reagents to purify these human liver P450 isozymes and to study their roles in polymorphic drug oxidation.l'2 Preparation of Highly Efficient Autoantibody Affinity Matrix P r i n c i p l e . An immunoaffinity matrix that essentially fully retains antibody potency can be prepared from human serum by absorbing the immunoglobulin G (IgG) fraction of whole serum to protein A-Sepharose (only IgG3 will not be bound). The antibody-protein A complex is then stabilized with a bifunctional cross-linking reagent, which prevents antibody leakage and further permits reuse of the antibody matrix. The specific interaction of protein A with the antibody Fc domain orients all antibodies in an optimal way for antigen binding so that essentially no loss of antibody potency owing to steric hindrance or antibody damage occurs. Procedure
I. Prepare a 30% stock suspension of protein A-Sepharose beads in 0.1 M sodium phosphate buffer, pH 7.4 (NaPi buffer). 2. Mix the serum sample (cleared by centrifugation or filtration) in a microcentrifuge tube with 3 volumes of the 30% stock suspension of protein A-Sepharose beads. Incubate at room temperature for at least 1 hr while gently rotating the tube. (One volume of protein A-Sepharose per volume of serum is sufficient to bind all IgGs.) 3. Spin in a microcentrifuge for 1 min and carefully aspirate the serum. Wash the pellet 3 times with 1 ml of NaPi buffer, the second wash buffer containing 1 M NaCI. (To avoid loss of beads, aspiration of the wash fluid is preferably done by using a narrowly drawn glass capillary attached to a water pump.) i U. M. Zanger, H. P. Hauri, J. Loeper, J. C. Homberg, and U. A. Meyer, Proc. Natl. Acad. Sci. U.S.A. 85, 8256 (1988). 2 U. Z. Meier and U. A. Meyer,Biochemistry 26, 8466 (1987).
U. A. Meyer,U. M. Zanger,D. Grant, and M. Blum,in "Advancesin Drug Research" (B. Testa, ed.), Vol. 19, p. 197. AcademicPress, London, 1990; (13) D. Sesardic, M. Pasanen, O. Pelkonen, and A. R. Boobis, Carcinogenesis 11, 1183 (1990); (14) K.-H. Meyer zum Buschenfelde,A. W. Lohse,M. Manns,and T. PoraUa,Hepatology 12, 354 (1990); (15) D. W. Nebert, D. R. Nelson, M. Adesnik,M. J. Coon, R. W. Estabrook, F. J. Gonzalez,F. P. Guengerich,I. C. Gunsalaus,E. F. Johnson;,B. Kemper,W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman,DNA 8, 1 (1989); (16) T. Yasumori, N. Murayama, Y. Yamazoe, and R. Kato, Clin. Pharmacol. Ther. 47, 313 (1990).
204
IMMUNOCHEMICAL METHODS
[20]
4. Cross-link antibodies to protein A by adding 3 volumes of 5% (v/v) glutaraldehyde (electron microscope grade) in NaPi buffer (other crosslinking reagents might be used instead of glutaraldehyde, following the instructions given by the manufacturer for reaction conditions). Incubate overnight with gentle mixing. 5. Wash the beads as described above. Block free aldehyde groups by incubation with 3 volumes of 0. I M ethanolamine (pH 7.4) for 4 hr to overnight at room temperature. 6. Wash the beads again extensively with NaP i buffer. The crosslinked antibody beads can be stored at 4 ° as a 30% stock suspension in NaPi buffer containing 0.1% sodium azide or merthiolate. Comments. The efficiency of the coupling reaction can be controlled by boiling 5-/~1 samples of beads taken before and after the coupling reaction in sodium dodecyl sulfate (SDS) sample buffer. Extracts are analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and staining of the gel with Coomassie blue. Antibody heavy and light chain bands should not be visible in extracts from cross-linked beads. If any leakage occurs, noncovalently bound antibodies can be removed by washing the beads with 0.1 M glycine (pH 3.0) followed by reequilibration in NaPi buffer. However, with anti-LKMl and anti-LKM2 antibody beads, no leakage was observed under a variety of elution conditions. The efficiency for antigen binding was tested by immunoprecipitation of the respective P450 activities and found to be comparable to antibodies noncovalently bound to protein A-Sepharose. 2 This technique of covalently coupling antibodies to protein A was originally described for rabbit antisera 3 (rabbit IgGs also bind with high affinity to protein A). A similar procedure can be used to cross-link monoclonal antibodies to protein A-Sepharose. 4
Immunoisolation of Microsomal Autoantigens Buffers and Solutions NaPi buffer: 0.1 M sodium phosphate buffer, pH 7.4 2x Solubilization buffer: 2% (w/v) Lubrol PX (or an equivalent detergent), 2% (w/v) sodium cholate, 40% (v/v) glycerol, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM phenylmethylsulfonyl fluoride (PMSF), and 2 mM dithiothreitol (DTT) in N a P i buffer 3 D. M. Gersten and J. J. Marchalonis, J. Immunol. Methods 24, 305 (1978). 4 C. Schneider, R. A. Newman, D. R. Sutherland, U. Asser, and M. F. Greaves, J. Biol. Chem. 257, 10766 (1982).
[20]
IMMUNOISOLATION OF P450 AUTOANTIGENS
205
1 x Solubilization buffer: 2× solubilization buffer diluted 1 : 1 with N a P i buffer 1/10x Solubilization buffer: I x solubilization buffer diluted 1 : 10 with water Washing buffer: 1 M KC1 in 1 x solubilization buffer
Solubilization and Antigen Binding 1. Mix microsomes (6 mg protein/ml in N a P i buffer) at a ratio of 1 : 1 with ice-cold, 2 × concentrated solubilization buffer and incubate on ice for 1 hr. 2. Pellet insoluble material by ultracentrifugation at 105,000 g for 1 hr. 3. In a 1.5-ml microcentrifuge tube, mix solubilized microsomes (0.1-1 mg protein in a volume of at least 0.2 ml of 1 x solubilization buffer) with 30/zl of antibody beads, which have been preequilibrated by washing with 1 ml of 1 × solubilization buffer. Incubate for at least 90 rain at 4 ° while rotating the tube. 4. Spin for 1 min and transfer the supernatant to a fresh tube (if required for further analysis). Wash the pellet with 1 ml of 1 x solubilization buffer and then at least 3 times with 1-ml portions of washing buffer, followed by a final wash cycle with 1 ml of 1/10× solubilization buffer. Aspirate as much liquid from the beads as possible. Bound proteins can now be eluted.
Elution of Antigen. An essentially quantitative control elution can be performed by extracting the washed beads at 95° consecutively with two 60-/zl aliquots of SDS sample buffer. Both extracts are combined and directly loaded on a 10% SDS-polyacrylamide gel. Essentially quantitative elution of LKM~ antigen isolated from human liver microsomes could also be achieved with two 60/xl aliquots of 0.1 M glycine (pH 3.0), containing 0.2% (w/v) Lubrol PX (at room temperature). The two extracts are pooled and should be neutralized by adding 5/zl of 1 M Tris base before electrophoresis is performed. Elution of LKM2 antigen without damaging the antibody matrix could only be achieved with 0.5% SDS in water as eluent. Several other elution conditions failed with LKM2 antibodies. 2 If reuse of the antibody beads is planned, these should be washed 3 times with l ml of elution buffer, followed by reequilibration in NaP i buffer, pH 7.4. Comments. Separation of the eluted proteins by SDS-PAGE and subsequent silver staining 5 of the gel reveal the apparent molecular weights and (under quantitative conditions) a rough estimation of the relative microsomal abundance of proteins, which bind with high affinity to the autoantibody. With a given sensitivity of silver staining of well below 50 5 C. R. Merril, D. Goldman, and M. L. Van Keuren, this series, Vol. 96, p. 230.
206
IMMUNOCHEMICALMETHODS
[20]
ng for a pure protein: a rare antigen of 0.01% of the total microsomal protein would still be visible, if isolated quantitatively from 0.5 mg of solubilized microsomes. However, in addition to the potential occurrence of heavy chain bands owing to antibody bleeding (see above), nonspecific adsorption of proteins to the matrix and nonpathological binding capacity of human serum IgG must be considered before conclusions are drawn. Control experiments should therefore be performed with a serum pool or with at least two sera from healthy individuals. Alternatively to silver staining, isolated proteins may be transferred from the gel to nitrocellulose and probed with antibodies of known specificity.6 Because the binding of antigen to the antibody is allowed to proceed under nondenaturing conditions, immunoisolation, unlike immunoblotting, is not restricted to antibodies recognizing denatured antigen. This was particularly important with anti-LKM 1 and anti-LKM2 antibodies, which often poorly react or even fail to react with SDS-denatured proteins on immunoblots.l,2,7'8 Example I: Semiquantitative Immunoisolation of P450IID6 from Human Liver Microsomes A selected anti-LKM~ serum was used for microscale immunoisolation of LKM~ antigen from solubilized microsomes of 17 different human livers (Fig. 1). The amount of antibody beads was sufficient to guarantee quantitative binding (>90%) of antigen in all samples. This had been previously tested by repeated incubations of one sample of solubilized microsomes with fresh aliquots of antibody beads. Three major protein bands with electrophoretic mobilities corresponding to relative molecular weights of 41,000, 50,000 and 55,000 were detected on a silver-stained gel. l In control experiments with sera from healthy individuals (not shown), only the broad band at 55K was observed, indicating that this protein(s) was nonspecifically bound to the antibody matrix. The 41K protein band did not show up with control serum, but was also absent with an anti-LKM~ serum from a different patient (not shown). The significance of this protein(s) is not known. The 50K protein band, however, was consistantly isolated with all anti-LKM~ sera tested. As shown in Fig. 1, the relative amounts of immunoisolated 50K protein were highly correlated to the P450IID66 A. Haid and M. Suissa, this series, Vol. 96, p. 192. 7 M. Manns, E. F. Johnson, K. J. Griffin, E. M. Tan, and K. F. Sullivan, J. Clin. Invest. 83, 1066 (1989). 8 D. J. Waxman, D. P. Lapenson, M. Krishnan, O. Bernard, G. Kreibich, and F. Alverez, Gastroenterology 95, 1326 (1988). 9 U. M. Zanger, F. Vilbois, J. Hardwick, and U. A. Meyer, Biochemistry 27, 5447 (1988).
[9.0]
IMMUNOISOLATION OF P450 AUTOANTIGENS
207
A 7~ 7~ 7~ "O "O "O "-I ~
liver
t-~ ~ "-,1 l0
I~ ~ W O
~
~
~1 ~1 ~ ~ ~ 0 1 1 ~ 01 ",1 ~
~
~
I~ ~ 1N 1 I~ I-,- ~,
I~ ~1 ~ I~ ~ 0 ~, ro ld .Ix LO ~- r~ c~
phenotype
B 100
"
0 0
_~
0
0
75-
Q
•o
50-
=
25-
% 0 •
rs = .96 p < .001 (n=17)
0 " ~
i
=
!
t
i
0
25
50
75
100
125
Vma x ( nmol l'-OH-buf / mg / 60 min )
FIG. 1. Comparison between relative amounts of immunoisolated LKMI antigen and microsomal P450IID6 activity in 17 human livers. (A) Immunoisolation was performed as described in the text with 0.5 mg of solubilized microsomai protein from each liver. Eluted proteins were separated by SDS-PAGE and visualized by silver staining. Of the 17 liver samples, 8 were obtained as wedge biopsies from individuals who had been phenotyped in oioo as extensive (EM) or poor metabolizers (PM) of debrisoquine [M. Eichelbaum, N. Spannbrucker, B. Steincke, and H. J. Dengler, Eur. J. Clin. Pharmacol. 16, 183 (1979)]. (B) The region of the silver-stained gel corresponding to a relative molecular mass of 50 kDa (arrow) was densitometrically analyzed. The resulting arbitrary density units were compared to the P450IID6 activities determined in intact microsomes of each liver. Activities are V=~x values for cumene hydroperoxide-mediated bufuralol l'-hydroxylation 9 (in nmol l'hydroxybufuralol formed/mg of protein/60 rain). Filled symbols represent data for biopsy samples from individuals with known phenotype ( | , PM; 0 , EM). All other livers are indicated by open symbols (©). The Spearman rank correlation coefficient rs was calculated from all data.
208
IMMUNOCHEMICAL METHODS
1 116
[20]
2
3
4
5
68
460
25
-
t
97
66
45
activity
FIG. 2. Purification of P450IID6 from human liver by anti-LKMl immunoaffinity chromatography. Solubilized human liver microsomes were first enriched for P450IID6 activity by anion-exchange chromatography on DEAE-cellulose. Aliquots of this fraction were then applied to a 0.6-ml anti-LKMJprotein A-Sepharose column. Loading of the column was continued until increasing P450IID6 activity was detected in the flow through. The column was extensively washed, and bound protein was eluted with 0.1 M glycine, pH 3.0. Aliquots from different fractions were analyzed by SDS-PAGE on a 10% gel, which was developed with silver. Lane 1, molecular weight (x 10 -3) marker proteins (0.5 /.tg each). Lane 2, solubilized human liver microsomes (5/xg). Lane 3, DEAE-ceflulose fraction (5/~g). Lane 4, anti-LKMl flow-through fraction (5 /~g). Lane 5, protein eluted from the anti-LKMl column, an amount corresponding to 2.5 mg of microsomal protein was applied. P450IID6 activities were determined in aliquots of the indicated fractions with 500/zM ( + )-bufuralol and 125 ~M cumene hydroperoxide 9 and are given as nmol l'-hydroxybufuralol/mg protein/ 60 min. [Reprinted from U. M. Zanger, H. P. Hauri, J. Loeper, J. C. Homberg, and U. A. Meyer, Proc. Natl. Acad. Sci. U.S.A. 88, 8256 (1988).]
catalyzed, microsomal activities for bufuralol l'-hydroxylation 9 in these livers. Furthermore, the 50K protein was not detectable in microsomes from several livers with very low P450IID6 activity. Among these were two liver biopsies obtained from in vivo phenotyped "poor metabolizer" (PM) individuals for debrisoquine, which lack P450IID6 protein, as had
[20l
IMMUNOISOLATION OF P450 AUTOANTIGENS
209
been found earlier with an antibody against rat P450dbl 9,~° (for review, see Meyer et al.ll).
Example II: Preparative Scale Immunoisolation of P450IID6 Immunoisolation of P450IID6 using L K M l antibodies was upscalcd and modified to obtain sufficientamounts of highly pure protein for Nterminal amino acid analysis and for the production of antibodies (Fig. 2). Because microscale experiments had indicated that sornc other proteins wcrc adsorbed to the L K M I immunoaffinity matrix in addition to P45011D6 (scc Fig. I),immunoisolation on a larger scale was performed on a prepurificd fraction of solubilizcd human liver microsomcs, which was approximately 7-fold enriched in P450IID6 activity. This fraction had bccn obtained by anion-exchange chromatography following P450IID6 activityin column cluates as described in detailcarlier.1Immunoaffinity chromatography was then performed on a small column containing 0.6 ml of L K M I antibody beads. Approximately 10/zg of a protein with an M r of 50K was obtained per 100 mg of microsomal protein, as judged by comparison to known amounts of molecular weight marker proteins on silver-stained polyacrylamidc gels (Fig. 2). Not surprisingly, only traces of enzymatic activity could bc recovered in the eluatcs. However, N-terminal amino acid sequencing confirrncd the identity of this protein with P45011D6.t L K M I thus provided a unique reagent by which P450IID6 could be isolated in an clcctrophorctically homogeneous form. Immunopurificd preparations of the L K M ~ and L K M 2 autoantigcns further proved to bc efficient antigens for the production of monoclonal and polyclonal antibodies, which in turn specificallyrecognized conventionally purified P45011D61 and P450mcph, 2 respectively. Acknowledgments Anti-LKM scra wcrc kindly provided by J. C. Hornbcrg (Paris, France). M. Eichclbaurn (Stuttgart, Gcrrnany), P. Beaunc (Paris, France), and W. Kalow and T. Inaba (Toronto, Canada) arc gratefully acknowledged for supplying liver samples. I thank U. A. Meyer (Basel, Switzerland) for helpful advice. This work was performed at the Department of Pharmacology, Bioccntcr, University of Basel (Switzerland), and was supported by Grant 3.817.87 of the Swiss National Science Foundation.
to F. J. Gonzalez, R. C. Skoda, S. Kirnura, M. Umeno, U. M. Zanger, D. W. Nebert, H. V. Gelboin, J. P. Hardwick, and U. A. Meyer, Nature (London) 331, 442 (1988). 11 U. A. Meyer, R. C. Skoda, and U. M. Zanger, Pharmacol. Ther. 46, 297 (1990)
IMMUNOISOLATION OF P 4 5 0 AUTOANTIGENS
201
mainly recognize native cytochromes P450,7 the antibodies directed against P450 fusion proteins recognize mainly denatured cytochromes P450. We were also successful in generating antibodies against P450 fusion proteins containing almost full-length P450IIC6 or P450IIC7 fusion protein. The antibodies recognized these proteins in microsomes. Thus, fusion proteins can be used to generate antibodies against a cytochrome P450 which is not available in a purified form in the laboratory. Acknowledgments We thank Mrs. I. Bfhm and Mrs. H. Steedfor typingthe manuscript.This work was supported by the DeutscheForschungsgemeinschaft(SFB 302).
[20] I m m u n o i s o l a t i o n of H u m a n M i c r o s o m a l C y t o c h r o m e s P 4 5 0 Using A u t o a n t i b o d i e s
By
ULRICH M.
ZANGER
Introduction Human cytochrome P450 isozymes of three P450 subfamilies have recently been shown to be specifically recognized by certain types of circulating autoantibodies, which occur in patients with either drug-induced or idiopathic chronic active forms of hepatitis (Table I; see also [21], this volume). All these P450 isozymes are mainly expressed in the liver, they metabolize xenobiotic compounds, and their catalytic function is potently inhibited by the respective autoantibodies. Besides the interest evoked by these autoantibodies in the mechanisms leading to autoimmunity and drug-induced hepatitis, they have proved to be highly valuable immunological tools of amazing specificity and potency. The protocols presented in this chapter describe the use of autoantisera to immunoisolate P450 proteins from solubilized microsomes. They may of course be applicable to other types of autoantibodies as well as to antibodies raised in rabbits. A microscale procedure is presented, which allows one to detect and semiquantitatively isolate microsomal antigens. By immunopurification on a larger scale, sufficient amounts of highly pure antigen can be obtained for further protein chemical analysis and for the production of antibodies. These methods have been used with the socalled anti-LKM (anti-liver/kidney microsome) antibodies type 1 and 2 to detect and identify their autoantigens, the liver microsomal P450IID6 and METHODS IN ENZYMOLOGY, VOL. 206
Copyright© 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.
202
IMMUNOCHEMICAL METHODS
[20]
TABLE I HUMAN P450 ISOZYMESRECOGNIZED BY AUTOANTIBODIES Autoantibody Anti-LKMI°
Anti-LKM2a
Anti-LMa Idiopathic chronic active (4) or druginduced (5) (dihydralazine) hepatitis P450IA2 (4), P450PA (5) IA2 Ethoxyresorufin (4,5), phenacetin
Liver disease
Autoimmune chronic active hepatitis type I~ (1,2)
Drug-induced (tienilic acid) hepatitis (3)
Recognized human P450 b P450 classifiedc Substrates
P450dbl (6-8), P450bufI (9) IID6 Debrisoquine, sparteine, bufuralol (12)
P450-8 (10), P450meph (11) IIC8/9/10 e Tienilic acid (10), mephenytoin (11)
Comments
Genetically polymorphicd
Genetically polymorphic e
(5) Inducible (4,13)
a Abbreviations are based on immunohistochemical classification: anti-LKMl~, anti-liver/ kidney microsome antibody type 1 (1,2) or 2 (3); anti-LM, antiliver microsome antibody (4,5). See Ref. (14) for a recent review. b Names of human P450 isozymes used by authors describing recognition by autoantibodies. c Classification according to the most recent proposal for cytochrome P450 nomenclature (15) P450IID6 has previously been designated as P450IID1 (12). d P450IID6 is not expressed in 5-10% of Caucasians [debrisoquine/sparteine type genetic polymorphism of drug oxidation; see Meyer et al. (12) for review]. e Anti-LKM2 possibly recognizes several closely related P450 isozymes of the same subfamily which are presently referred to as P450IIC8/9 (12) or P450IIC8D/10 (5). One of these or a highly similar isozyme appears to be affected by the mephenytoin type genetic polymorphism (11,12,16). f Key to references: (1) M. Rizzetto, G. Swana, and D. Doniach, Clin. Exp. lmmunol. 15, 331 (1973); (2) J. C. Homberg, N. Abuaf, O. Bernard, S. Islam, F. Alvarez, S. H. Khalil, R. Poupon, F. Darnis, V.-G. Levy, P. Grippon, P. Opolon, J. Beruuau, J.-P. Benhamou, and D. Alagille, Hepatology 7, 1333 (1987); (3) J.-C. Homberg, C. Andre, and N. Abuaf, Clin. Exp. Immunol. 55, 561 (1984); (4) M. P. Manns, K. J. Griffin, L. C. Quattrochi, M. Sacher, H. Thaler, R. H. Tukey, and E. F. Johnson, Arch. Biochem. Biophys. 7,80, 229 (1990). (5) M. Bourdi, D. Larrey, J. Nataf, J. Bernuau, D. Pessayre, M. Iwasalli, F. P. Guengerich, and P. H. Beaune, J. Clin. Invest. 85, 1967 (1990); (6) U. M. Zanger, H. P. Hauri, J. Loeper, J. C. Homberg, and U. A. Meyer, Proc. Natl. Acad. Sci. U.S.A. 85, 8256 (1988); (7) M. Manns, E. F. Johnson, K. J. Griffin, E. M. Tan, and K. F. Sullivan, J. Clin. Invest. 83, 1066 (1989); (8) M. Gueguen, A. M. Yamamoto, O. Benard, and F. Alvarez, Biochem. Biophys. Res. Commun. 159, 542 (1989); (9) L. Kiffel, J. Loeper, J. C. Homberg, and J. P. Leroux, Biochem. Biophys. Res. Commun. 159, 283 (1989); (10) P. Beaune, P. M. Dansette, D. Mansuy, L. Kiffel, M. Finck, C. Amar, J. P. Leroux, and J. C. Homberg, Proc. Natl. Acad. Sci. U.S.A. 84, 551 (1987); (11) U. T. Meier and U. A. Meyer, Biochemistry 26, 8466 (1987); (12)
[20]
IMMUNOISOLATION OF P450 AUTOANTIGENS
203
P450meph, respectively (Table I). Anti-LKM antibodies provided unique reagents to purify these human liver P450 isozymes and to study their roles in polymorphic drug oxidation.l'2 Preparation of Highly Efficient Autoantibody Affinity Matrix P r i n c i p l e . An immunoaffinity matrix that essentially fully retains antibody potency can be prepared from human serum by absorbing the immunoglobulin G (IgG) fraction of whole serum to protein A-Sepharose (only IgG3 will not be bound). The antibody-protein A complex is then stabilized with a bifunctional cross-linking reagent, which prevents antibody leakage and further permits reuse of the antibody matrix. The specific interaction of protein A with the antibody Fc domain orients all antibodies in an optimal way for antigen binding so that essentially no loss of antibody potency owing to steric hindrance or antibody damage occurs. Procedure
I. Prepare a 30% stock suspension of protein A-Sepharose beads in 0.1 M sodium phosphate buffer, pH 7.4 (NaPi buffer). 2. Mix the serum sample (cleared by centrifugation or filtration) in a microcentrifuge tube with 3 volumes of the 30% stock suspension of protein A-Sepharose beads. Incubate at room temperature for at least 1 hr while gently rotating the tube. (One volume of protein A-Sepharose per volume of serum is sufficient to bind all IgGs.) 3. Spin in a microcentrifuge for 1 min and carefully aspirate the serum. Wash the pellet 3 times with 1 ml of NaPi buffer, the second wash buffer containing 1 M NaCI. (To avoid loss of beads, aspiration of the wash fluid is preferably done by using a narrowly drawn glass capillary attached to a water pump.) i U. M. Zanger, H. P. Hauri, J. Loeper, J. C. Homberg, and U. A. Meyer, Proc. Natl. Acad. Sci. U.S.A. 85, 8256 (1988). 2 U. Z. Meier and U. A. Meyer,Biochemistry 26, 8466 (1987).
U. A. Meyer,U. M. Zanger,D. Grant, and M. Blum,in "Advancesin Drug Research" (B. Testa, ed.), Vol. 19, p. 197. AcademicPress, London, 1990; (13) D. Sesardic, M. Pasanen, O. Pelkonen, and A. R. Boobis, Carcinogenesis 11, 1183 (1990); (14) K.-H. Meyer zum Buschenfelde,A. W. Lohse,M. Manns,and T. PoraUa,Hepatology 12, 354 (1990); (15) D. W. Nebert, D. R. Nelson, M. Adesnik,M. J. Coon, R. W. Estabrook, F. J. Gonzalez,F. P. Guengerich,I. C. Gunsalaus,E. F. Johnson;,B. Kemper,W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman,DNA 8, 1 (1989); (16) T. Yasumori, N. Murayama, Y. Yamazoe, and R. Kato, Clin. Pharmacol. Ther. 47, 313 (1990).
204
IMMUNOCHEMICAL METHODS
[20]
4. Cross-link antibodies to protein A by adding 3 volumes of 5% (v/v) glutaraldehyde (electron microscope grade) in NaPi buffer (other crosslinking reagents might be used instead of glutaraldehyde, following the instructions given by the manufacturer for reaction conditions). Incubate overnight with gentle mixing. 5. Wash the beads as described above. Block free aldehyde groups by incubation with 3 volumes of 0. I M ethanolamine (pH 7.4) for 4 hr to overnight at room temperature. 6. Wash the beads again extensively with NaP i buffer. The crosslinked antibody beads can be stored at 4 ° as a 30% stock suspension in NaPi buffer containing 0.1% sodium azide or merthiolate. Comments. The efficiency of the coupling reaction can be controlled by boiling 5-/~1 samples of beads taken before and after the coupling reaction in sodium dodecyl sulfate (SDS) sample buffer. Extracts are analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and staining of the gel with Coomassie blue. Antibody heavy and light chain bands should not be visible in extracts from cross-linked beads. If any leakage occurs, noncovalently bound antibodies can be removed by washing the beads with 0.1 M glycine (pH 3.0) followed by reequilibration in NaPi buffer. However, with anti-LKMl and anti-LKM2 antibody beads, no leakage was observed under a variety of elution conditions. The efficiency for antigen binding was tested by immunoprecipitation of the respective P450 activities and found to be comparable to antibodies noncovalently bound to protein A-Sepharose. 2 This technique of covalently coupling antibodies to protein A was originally described for rabbit antisera 3 (rabbit IgGs also bind with high affinity to protein A). A similar procedure can be used to cross-link monoclonal antibodies to protein A-Sepharose. 4
Immunoisolation of Microsomal Autoantigens Buffers and Solutions NaPi buffer: 0.1 M sodium phosphate buffer, pH 7.4 2x Solubilization buffer: 2% (w/v) Lubrol PX (or an equivalent detergent), 2% (w/v) sodium cholate, 40% (v/v) glycerol, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM phenylmethylsulfonyl fluoride (PMSF), and 2 mM dithiothreitol (DTT) in N a P i buffer 3 D. M. Gersten and J. J. Marchalonis, J. Immunol. Methods 24, 305 (1978). 4 C. Schneider, R. A. Newman, D. R. Sutherland, U. Asser, and M. F. Greaves, J. Biol. Chem. 257, 10766 (1982).
[20]
IMMUNOISOLATION OF P450 AUTOANTIGENS
205
1 x Solubilization buffer: 2× solubilization buffer diluted 1 : 1 with N a P i buffer 1/10x Solubilization buffer: I x solubilization buffer diluted 1 : 10 with water Washing buffer: 1 M KC1 in 1 x solubilization buffer
Solubilization and Antigen Binding 1. Mix microsomes (6 mg protein/ml in N a P i buffer) at a ratio of 1 : 1 with ice-cold, 2 × concentrated solubilization buffer and incubate on ice for 1 hr. 2. Pellet insoluble material by ultracentrifugation at 105,000 g for 1 hr. 3. In a 1.5-ml microcentrifuge tube, mix solubilized microsomes (0.1-1 mg protein in a volume of at least 0.2 ml of 1 x solubilization buffer) with 30/zl of antibody beads, which have been preequilibrated by washing with 1 ml of 1 × solubilization buffer. Incubate for at least 90 rain at 4 ° while rotating the tube. 4. Spin for 1 min and transfer the supernatant to a fresh tube (if required for further analysis). Wash the pellet with 1 ml of 1 x solubilization buffer and then at least 3 times with 1-ml portions of washing buffer, followed by a final wash cycle with 1 ml of 1/10× solubilization buffer. Aspirate as much liquid from the beads as possible. Bound proteins can now be eluted.
Elution of Antigen. An essentially quantitative control elution can be performed by extracting the washed beads at 95° consecutively with two 60-/zl aliquots of SDS sample buffer. Both extracts are combined and directly loaded on a 10% SDS-polyacrylamide gel. Essentially quantitative elution of LKM~ antigen isolated from human liver microsomes could also be achieved with two 60/xl aliquots of 0.1 M glycine (pH 3.0), containing 0.2% (w/v) Lubrol PX (at room temperature). The two extracts are pooled and should be neutralized by adding 5/zl of 1 M Tris base before electrophoresis is performed. Elution of LKM2 antigen without damaging the antibody matrix could only be achieved with 0.5% SDS in water as eluent. Several other elution conditions failed with LKM2 antibodies. 2 If reuse of the antibody beads is planned, these should be washed 3 times with l ml of elution buffer, followed by reequilibration in NaP i buffer, pH 7.4. Comments. Separation of the eluted proteins by SDS-PAGE and subsequent silver staining 5 of the gel reveal the apparent molecular weights and (under quantitative conditions) a rough estimation of the relative microsomal abundance of proteins, which bind with high affinity to the autoantibody. With a given sensitivity of silver staining of well below 50 5 C. R. Merril, D. Goldman, and M. L. Van Keuren, this series, Vol. 96, p. 230.
206
IMMUNOCHEMICALMETHODS
[20]
ng for a pure protein: a rare antigen of 0.01% of the total microsomal protein would still be visible, if isolated quantitatively from 0.5 mg of solubilized microsomes. However, in addition to the potential occurrence of heavy chain bands owing to antibody bleeding (see above), nonspecific adsorption of proteins to the matrix and nonpathological binding capacity of human serum IgG must be considered before conclusions are drawn. Control experiments should therefore be performed with a serum pool or with at least two sera from healthy individuals. Alternatively to silver staining, isolated proteins may be transferred from the gel to nitrocellulose and probed with antibodies of known specificity.6 Because the binding of antigen to the antibody is allowed to proceed under nondenaturing conditions, immunoisolation, unlike immunoblotting, is not restricted to antibodies recognizing denatured antigen. This was particularly important with anti-LKM 1 and anti-LKM2 antibodies, which often poorly react or even fail to react with SDS-denatured proteins on immunoblots.l,2,7'8 Example I: Semiquantitative Immunoisolation of P450IID6 from Human Liver Microsomes A selected anti-LKM~ serum was used for microscale immunoisolation of LKM~ antigen from solubilized microsomes of 17 different human livers (Fig. 1). The amount of antibody beads was sufficient to guarantee quantitative binding (>90%) of antigen in all samples. This had been previously tested by repeated incubations of one sample of solubilized microsomes with fresh aliquots of antibody beads. Three major protein bands with electrophoretic mobilities corresponding to relative molecular weights of 41,000, 50,000 and 55,000 were detected on a silver-stained gel. l In control experiments with sera from healthy individuals (not shown), only the broad band at 55K was observed, indicating that this protein(s) was nonspecifically bound to the antibody matrix. The 41K protein band did not show up with control serum, but was also absent with an anti-LKM~ serum from a different patient (not shown). The significance of this protein(s) is not known. The 50K protein band, however, was consistantly isolated with all anti-LKM~ sera tested. As shown in Fig. 1, the relative amounts of immunoisolated 50K protein were highly correlated to the P450IID66 A. Haid and M. Suissa, this series, Vol. 96, p. 192. 7 M. Manns, E. F. Johnson, K. J. Griffin, E. M. Tan, and K. F. Sullivan, J. Clin. Invest. 83, 1066 (1989). 8 D. J. Waxman, D. P. Lapenson, M. Krishnan, O. Bernard, G. Kreibich, and F. Alverez, Gastroenterology 95, 1326 (1988). 9 U. M. Zanger, F. Vilbois, J. Hardwick, and U. A. Meyer, Biochemistry 27, 5447 (1988).
[9.0]
IMMUNOISOLATION OF P450 AUTOANTIGENS
207
A 7~ 7~ 7~ "O "O "O "-I ~
liver
t-~ ~ "-,1 l0
I~ ~ W O
~
~
~1 ~1 ~ ~ ~ 0 1 1 ~ 01 ",1 ~
~
~
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I~ ~1 ~ I~ ~ 0 ~, ro ld .Ix LO ~- r~ c~
phenotype
B 100
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0 0
_~
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Q
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rs = .96 p < .001 (n=17)
0 " ~
i
=
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i
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25
50
75
100
125
Vma x ( nmol l'-OH-buf / mg / 60 min )
FIG. 1. Comparison between relative amounts of immunoisolated LKMI antigen and microsomal P450IID6 activity in 17 human livers. (A) Immunoisolation was performed as described in the text with 0.5 mg of solubilized microsomai protein from each liver. Eluted proteins were separated by SDS-PAGE and visualized by silver staining. Of the 17 liver samples, 8 were obtained as wedge biopsies from individuals who had been phenotyped in oioo as extensive (EM) or poor metabolizers (PM) of debrisoquine [M. Eichelbaum, N. Spannbrucker, B. Steincke, and H. J. Dengler, Eur. J. Clin. Pharmacol. 16, 183 (1979)]. (B) The region of the silver-stained gel corresponding to a relative molecular mass of 50 kDa (arrow) was densitometrically analyzed. The resulting arbitrary density units were compared to the P450IID6 activities determined in intact microsomes of each liver. Activities are V=~x values for cumene hydroperoxide-mediated bufuralol l'-hydroxylation 9 (in nmol l'hydroxybufuralol formed/mg of protein/60 rain). Filled symbols represent data for biopsy samples from individuals with known phenotype ( | , PM; 0 , EM). All other livers are indicated by open symbols (©). The Spearman rank correlation coefficient rs was calculated from all data.
208
IMMUNOCHEMICAL METHODS
1 116
[20]
2
3
4
5
68
460
25
-
t
97
66
45
activity
FIG. 2. Purification of P450IID6 from human liver by anti-LKMl immunoaffinity chromatography. Solubilized human liver microsomes were first enriched for P450IID6 activity by anion-exchange chromatography on DEAE-cellulose. Aliquots of this fraction were then applied to a 0.6-ml anti-LKMJprotein A-Sepharose column. Loading of the column was continued until increasing P450IID6 activity was detected in the flow through. The column was extensively washed, and bound protein was eluted with 0.1 M glycine, pH 3.0. Aliquots from different fractions were analyzed by SDS-PAGE on a 10% gel, which was developed with silver. Lane 1, molecular weight (x 10 -3) marker proteins (0.5 /.tg each). Lane 2, solubilized human liver microsomes (5/xg). Lane 3, DEAE-ceflulose fraction (5/~g). Lane 4, anti-LKMl flow-through fraction (5 /~g). Lane 5, protein eluted from the anti-LKMl column, an amount corresponding to 2.5 mg of microsomal protein was applied. P450IID6 activities were determined in aliquots of the indicated fractions with 500/zM ( + )-bufuralol and 125 ~M cumene hydroperoxide 9 and are given as nmol l'-hydroxybufuralol/mg protein/ 60 min. [Reprinted from U. M. Zanger, H. P. Hauri, J. Loeper, J. C. Homberg, and U. A. Meyer, Proc. Natl. Acad. Sci. U.S.A. 88, 8256 (1988).]
catalyzed, microsomal activities for bufuralol l'-hydroxylation 9 in these livers. Furthermore, the 50K protein was not detectable in microsomes from several livers with very low P450IID6 activity. Among these were two liver biopsies obtained from in vivo phenotyped "poor metabolizer" (PM) individuals for debrisoquine, which lack P450IID6 protein, as had
[20l
IMMUNOISOLATION OF P450 AUTOANTIGENS
209
been found earlier with an antibody against rat P450dbl 9,~° (for review, see Meyer et al.ll).
Example II: Preparative Scale Immunoisolation of P450IID6 Immunoisolation of P450IID6 using L K M l antibodies was upscalcd and modified to obtain sufficientamounts of highly pure protein for Nterminal amino acid analysis and for the production of antibodies (Fig. 2). Because microscale experiments had indicated that sornc other proteins wcrc adsorbed to the L K M I immunoaffinity matrix in addition to P45011D6 (scc Fig. I),immunoisolation on a larger scale was performed on a prepurificd fraction of solubilizcd human liver microsomcs, which was approximately 7-fold enriched in P450IID6 activity. This fraction had bccn obtained by anion-exchange chromatography following P450IID6 activityin column cluates as described in detailcarlier.1Immunoaffinity chromatography was then performed on a small column containing 0.6 ml of L K M I antibody beads. Approximately 10/zg of a protein with an M r of 50K was obtained per 100 mg of microsomal protein, as judged by comparison to known amounts of molecular weight marker proteins on silver-stained polyacrylamidc gels (Fig. 2). Not surprisingly, only traces of enzymatic activity could bc recovered in the eluatcs. However, N-terminal amino acid sequencing confirrncd the identity of this protein with P45011D6.t L K M I thus provided a unique reagent by which P450IID6 could be isolated in an clcctrophorctically homogeneous form. Immunopurificd preparations of the L K M ~ and L K M 2 autoantigcns further proved to bc efficient antigens for the production of monoclonal and polyclonal antibodies, which in turn specificallyrecognized conventionally purified P45011D61 and P450mcph, 2 respectively. Acknowledgments Anti-LKM scra wcrc kindly provided by J. C. Hornbcrg (Paris, France). M. Eichclbaurn (Stuttgart, Gcrrnany), P. Beaunc (Paris, France), and W. Kalow and T. Inaba (Toronto, Canada) arc gratefully acknowledged for supplying liver samples. I thank U. A. Meyer (Basel, Switzerland) for helpful advice. This work was performed at the Department of Pharmacology, Bioccntcr, University of Basel (Switzerland), and was supported by Grant 3.817.87 of the Swiss National Science Foundation.
to F. J. Gonzalez, R. C. Skoda, S. Kirnura, M. Umeno, U. M. Zanger, D. W. Nebert, H. V. Gelboin, J. P. Hardwick, and U. A. Meyer, Nature (London) 331, 442 (1988). 11 U. A. Meyer, R. C. Skoda, and U. M. Zanger, Pharmacol. Ther. 46, 297 (1990)
