ARCHIVES
OF BIOCHEMISTRY
Vol. 291, No. 1, November
AND
BIOPHYSICS
15, pp. 168-175,
1991
Rat Sulfite Oxidase Antibodies Cross-React with Two Gene Family-Related Proteins: Albumin and Vitamin D-Binding Protein’ Daniel
B. Bellissimo and K. V. Rajagopalan2 of Biochemistry, Duke University Medical Center, Durham,
Department
Received
June
10, 1991, and in revised
form
July
North
Carolina
27710
29, 1991
Screening X cDNA libraries from rat liver with antibody to native rat liver sulfite oxidase (RLSO) showed cross-reaction with two proteins that belong to the same gene family: serum albumin and vitamin D-binding protein. Antibodies raised against native RLSO or sodium dodecyl sulfate-denatured protein cross-reacted with these proteins by Western blot analysis. The relative effectiveness of RLSO antibody binding was estimated to be l/5 for rat serum albumin and l/l0 for rat vitamin D-binding protein. This result was not caused by contaminating proteins in the RLSO used for immunization as the RLSO preparation did not react with rat serum albumin antibody. RLSO antibodies, selected for their ability to bind rat serum albumin immobilized on nitrocellulose, recognized both rat serum albumin and RLSO. RLSO antibody, with albumin-reactive antibody removed, still recognized vitamin D-binding protein, suggesting that multiple determinants specific to each protein are involved in the cross-reaction. Comparison of RLSO antibody binding to the rat and human proteins indicated that the determinants were species-specific. cDNA clones identified by screening cDNA libraries with RLSO antibody demonstrated that these determinants reside in the C-terminal domain of these proteins. These results suggest that these proteins contain some common immunological features and may be evolutionarily related. o 1991 AC&& preps, ha.
Rat liver sulfite oxidase (RLS0)3 is a molybdoenzyme located in the intermembrane spaceof mitochondria. The 1 This work was supported by NIH Grant GM 00091. D.B.B. was the recipient of a JB Duke Fellowship from Duke University. ’ To whom correspondence should be addressed. 3 Abbreviations used: RLSO, rat liver sulfite oxidase; RSA, rat serum albumin; RDBP, rat vitamin D-binding protein; DBP, vitamin D-binding protein; G,, group-specific component; D’M’, dithiothreitoh SDS, sodium
enzyme catalyzes the terminal step in the degradation of sulfur containing amino acids: the oxidation of sulfite to sulfate. It is a dimer of identical subunits of 58,000 daltons. Each subunit contains a molybdopterin and a cytochrome b5 in structurally distinct domains (1). In order to identify the amino acid residuesplaying a crucial role in enzymatic activity and molybdopterin binding, we attempted to clone RLSO using the X gtll system and immunoscreening with antibody to RLSO. The majority of the clones isolated encoded rat serum albumin (RSA) and rat vitamin Dbinding protein (RDBP). Although cloning of unrelated proteins is not uncommon when screening cDNA libraries with antibodies, our finding was unusual in that both proteins are members of the same gene family. Albumin, vitamin D-binding protein (DBP), and a-fetoprotein are all major serum proteins synthesized by mammalian liver and belong to the same gene family (24). There is 24 and 33% amino acid homology, and 41 and 48% nucleic acid homology between albumin and vitamin D-binding protein or a-fetoprotein, respectively. There is remarkable conservation of cysteines which are responsible for the stable domain structure in each of the three internally homologous domains. The domain structure of these proteins is also represented in the exonintron structure of their genes (3, 5, 6). Although these proteins have structural and evolutionary similarities, albumin and cr-fetoprotein have different functions from that of DBP. Albumin binds and transports long chain fatty acids, binds and detoxifies bilirubin, and binds indoles, CU+~,and NF2 (7). It also functions as a backup transport system of thyroid hormones, steroid hormones, tryptophan, and vitamin D sterols (8, 9). Albumin, though involved in a number of functions, is not dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TBS buffer, 0.05 M Tris-HCl, pH 8.0, 0.15 M NaCl; BSA, bovine serum albumin; HLSO, human liver sulfite oxidase, HSA, human serum albumin; HDBP, human vitamin D-binding protein.
168 All
Copyright 0 1991 rights of reproduction
0003.9S61/91 $3.00 by Academic Press, Inc. in any form reserved.
SULFITE
OXIDASE
ANTIBODIES:
CROSS-REACTION
essential for viability as there are examples of analbuminemia in humans and rats (10, 11). DBP, also called group-specific component (G,), was originally identified as a polymorphic marker in the CCglobulin region (12). The protein serves to transport vitamin D and its hydroxylated derivatives (13). The DBP concentration in serum is in marked excess over that required for vitamin D sterol transport alone. DBP binds to monomers of actin with high affinity (14) thereby sequestering potentially harmful actin filaments from the blood. DBP has also been found to associate with membrane-bound immunoglobulin on the surface of B lymphocytes (15) and with the IgG F, receptor on the membranes of a subpopulation of T cells (16). There has been extensive screening of the human DBP/G, polymorphisms and no G,O homozygotes have been detected (17). This suggests that some or all of DBP’s functions are critical for survival. Despite the structural similarities of albumin, a-fetoprotein, and vitamin D-binding protein, the native proteins are not immunologically cross-reactive. However, antisera to reduced and carboxymethylated RSA or rat cr-fetoprotein are cross-reactive with all three proteins (18). The cross-reactive antibodies appear to recognize sequential determinants in the primary structure that are not exposed during immunization with the native protein. RDBP showed weaker immunological cross-reactivity than RSA or rat a-fetoprotein. In the present studies immunoscreening of cDNA libraries suggested that RLSO is antigenically related to these gene family-related proteins. Given the functional diversity within this family, it was conceivable that sulfite oxidase could be related to them in some way. In order to address this point, we used Western blot and dot blot analyses to examine the cross-reaction of RLSO antibodies to RSA and RDBP and to characterize the determinants involved. MATERIALS
AND
METHODS
Materials. Electrophoresis materials, nitrocellulose, and Affi-Gel15 were purchased from Bio-Rad. “‘I-protein A (>30 or >70 pCi/ng) was from ICN. Freund’s adjuvant was from Difco. Rabbit skeletal muscle was from Pel-Freeze. Rabbit anti-rat albumin antibody was purchased from Cooper Labs. Rat albumin was purchased from Sigma. The material contained a single band as judged by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Protein A-Sepharose 4B and all other biochemicals were purchased from Sigma. Human vitamin Dbinding protein was kindly provided by Dr. John Haddad (Univ. of Pennsylvania). Sulfite oxidasepur$ication. Rat and human sulfite oxidase proteins were purified according to the procedure of Johnson and Rajagopalan (19). To ensure that RSA and RDBP did not contaminate the rat preparation, the rat livers were perfused with ice cold 0.25 M sucrose prior to homogenization to remove as much blood as possible. Purification steps were monitored for RSA with RSA antibody. DEAE-cellulose fractions with the best A,JAzao ratios were pooled, concentrated by Amicon filtration with a PM10 membrane, and fractionated by FPLC (Pharmacia) on a preparative Superose-12 column run in 0.05 M TrisHCl, pH 8.0,O.l mM EDTA. Fractions with AdJAm > 0.8 were further
WITH
RELATED
PROTEINS
169
purified by FPLC Mono Q chromatography using a 30-ml gradient of 0.2-0.45 M NaCl. Fractions with A4,,/Aw = 1.0 were isolated. By all criteria established previously, this is pure enzyme. Sulfite oxidase activity was stained in gels according to the procedure of Cohen (20). DEP purification. Rat DBP was purified by actin affinity chromatography (21). Afli-Gel-15 (25 ml) was washed as described and incubated overnight at 4’C with 25 ml G-actin (5 mg/ml) prepared from frozen rabbit skeletal muscle as described by Pardee and Spudich (22). The gel was extensively washed in depolymerizing buffer (0.01 M Tris-HCl, pH 8.0, 0.2 mM CaCl,, 0.5 mM NasATP, 1 mM NaNs, 0.2 mM DTT). The gel was used within 48 h of its preparation. Rat blood was collected from decapitated animals in the presence of 2 mM EDTA. The red cells were removed by centrifugation at 4000g. Serum was stored at -20°C until use. Serum was thawed, then centrifuged to remove particulate matter before application to the actin affinity column. Serum (20-40 ml) was passed through the column twice before washing and eluting as described. The protein from actin affinity chromatography was further purified by FPLC Mono Q chromatography. The sample in 0.01 M Tris-HCl, pH 8.0, was loaded, then washed with 0.05 M Tris-HCl, pH 8.0. DBP was eluted with a 30-ml gradient of O-O.4 M NaCl. The purity was estimated to be >90% by SDS-polyacrylamide electrophoresis (SDSPAGE). SDS-polyacrylamide gel ekctrophoresis and Western blot analysis. SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (23) in a Bio-Rad Protean electrophoresis unit. Transfer of protein bands from SDS-polyacrylamide gels to nitrocellulose by the LKB Novablot semidry blotter was effected according to the manufacturer’s instructions. Transfer to nitrocellulose paper was done at 0.8 mA/cmx for 1.5 h. The paper was washed in TBS (0.05 M Tris-HCl, pH 8.0, 0.15 M NaCl) then incubated in 5% nonfat dry milk in 0.5X TBS for l-3 h. Incubation with the antibody was carried out in a Seal-AMeal bag in the presence of the blocking agent (2% BSA or milk) at 4°C overnight. A 5% milk solution contains approximately 0.1 mg/ml BSA (24). One-hundred micrograms of IgG was typically used per blot. The blot was washed three times with TBS for 15 min each. The second wash contained 0.1% TX-100. The blot was incubated in a bag with 1.0 pCi ‘?-protein A in TBS with the blocking agent for 3-6 h. The blot was washed as described above and then autoradiographed on Kodak X-OMAT AR film. Dot blots were carried out by spotting protein solution on a nitrocellulose filter. The paper was allowed to dry. All further incubations were as described for Western blots. Immunization protocol. A New Zealand white rabbit was injected subcutaneously at multiple sites with 400 pg RLSO in Freund’s complete adjuvant. One month later a booster injection of 800 pg was given in incomplete adjuvant. The rabbit was bled 1 week later. Antibody to SDS-denatured homogeneous RLSO was prepared by the following method. Approximately 200 pg of protein was subjected to electrophoresis in 7.5% SDS-polyacrylamide gel. The gel was briefly stained with Coomassie blue R-250 so that the location of the RLSO could be identified, then destained. The gel was soaked in water, then the protein bands were cut out with a razor blade. The segment was mashed, then homogenized with 1.5 ml of water and 1.5 ml of complete adjuvant. The emulsion was injected subcutaneously at multiple sites in a New Zealand white rabbit. The polyacrylamide also acts as an adjuvant in this case (25). One month later, a booster injection of 160 pg of the antigen in incomplete adjuvant was given. The rabbit was bled from the ear 7 days after the booster injection. ZgG preparation. Blood was allowed to clot at 4“C overnight. The clot was centrifuged at 4000 rpm in a SS34 rotor for 15 min. The serum was removed and stored at -20°C until needed. Protein A-Sepharose (5 ml) was equilibrated with 50 mM Tris-HCl, pH 8.0,0.15 M NaCl, 0.1 mM EDTA. One milliliter of rabbit serum was passed over the column and rinsed with 1 column vol of buffer. The unbound material was passed over the column a second time. The column was washed until the A,, of the elute was
OF BIOCHEMISTRY
Vol. 291, No. 1, November
AND
BIOPHYSICS
15, pp. 168-175,
1991
Rat Sulfite Oxidase Antibodies Cross-React with Two Gene Family-Related Proteins: Albumin and Vitamin D-Binding Protein’ Daniel
B. Bellissimo and K. V. Rajagopalan2 of Biochemistry, Duke University Medical Center, Durham,
Department
Received
June
10, 1991, and in revised
form
July
North
Carolina
27710
29, 1991
Screening X cDNA libraries from rat liver with antibody to native rat liver sulfite oxidase (RLSO) showed cross-reaction with two proteins that belong to the same gene family: serum albumin and vitamin D-binding protein. Antibodies raised against native RLSO or sodium dodecyl sulfate-denatured protein cross-reacted with these proteins by Western blot analysis. The relative effectiveness of RLSO antibody binding was estimated to be l/5 for rat serum albumin and l/l0 for rat vitamin D-binding protein. This result was not caused by contaminating proteins in the RLSO used for immunization as the RLSO preparation did not react with rat serum albumin antibody. RLSO antibodies, selected for their ability to bind rat serum albumin immobilized on nitrocellulose, recognized both rat serum albumin and RLSO. RLSO antibody, with albumin-reactive antibody removed, still recognized vitamin D-binding protein, suggesting that multiple determinants specific to each protein are involved in the cross-reaction. Comparison of RLSO antibody binding to the rat and human proteins indicated that the determinants were species-specific. cDNA clones identified by screening cDNA libraries with RLSO antibody demonstrated that these determinants reside in the C-terminal domain of these proteins. These results suggest that these proteins contain some common immunological features and may be evolutionarily related. o 1991 AC&& preps, ha.
Rat liver sulfite oxidase (RLS0)3 is a molybdoenzyme located in the intermembrane spaceof mitochondria. The 1 This work was supported by NIH Grant GM 00091. D.B.B. was the recipient of a JB Duke Fellowship from Duke University. ’ To whom correspondence should be addressed. 3 Abbreviations used: RLSO, rat liver sulfite oxidase; RSA, rat serum albumin; RDBP, rat vitamin D-binding protein; DBP, vitamin D-binding protein; G,, group-specific component; D’M’, dithiothreitoh SDS, sodium
enzyme catalyzes the terminal step in the degradation of sulfur containing amino acids: the oxidation of sulfite to sulfate. It is a dimer of identical subunits of 58,000 daltons. Each subunit contains a molybdopterin and a cytochrome b5 in structurally distinct domains (1). In order to identify the amino acid residuesplaying a crucial role in enzymatic activity and molybdopterin binding, we attempted to clone RLSO using the X gtll system and immunoscreening with antibody to RLSO. The majority of the clones isolated encoded rat serum albumin (RSA) and rat vitamin Dbinding protein (RDBP). Although cloning of unrelated proteins is not uncommon when screening cDNA libraries with antibodies, our finding was unusual in that both proteins are members of the same gene family. Albumin, vitamin D-binding protein (DBP), and a-fetoprotein are all major serum proteins synthesized by mammalian liver and belong to the same gene family (24). There is 24 and 33% amino acid homology, and 41 and 48% nucleic acid homology between albumin and vitamin D-binding protein or a-fetoprotein, respectively. There is remarkable conservation of cysteines which are responsible for the stable domain structure in each of the three internally homologous domains. The domain structure of these proteins is also represented in the exonintron structure of their genes (3, 5, 6). Although these proteins have structural and evolutionary similarities, albumin and cr-fetoprotein have different functions from that of DBP. Albumin binds and transports long chain fatty acids, binds and detoxifies bilirubin, and binds indoles, CU+~,and NF2 (7). It also functions as a backup transport system of thyroid hormones, steroid hormones, tryptophan, and vitamin D sterols (8, 9). Albumin, though involved in a number of functions, is not dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TBS buffer, 0.05 M Tris-HCl, pH 8.0, 0.15 M NaCl; BSA, bovine serum albumin; HLSO, human liver sulfite oxidase, HSA, human serum albumin; HDBP, human vitamin D-binding protein.
168 All
Copyright 0 1991 rights of reproduction
0003.9S61/91 $3.00 by Academic Press, Inc. in any form reserved.
SULFITE
OXIDASE
ANTIBODIES:
CROSS-REACTION
essential for viability as there are examples of analbuminemia in humans and rats (10, 11). DBP, also called group-specific component (G,), was originally identified as a polymorphic marker in the CCglobulin region (12). The protein serves to transport vitamin D and its hydroxylated derivatives (13). The DBP concentration in serum is in marked excess over that required for vitamin D sterol transport alone. DBP binds to monomers of actin with high affinity (14) thereby sequestering potentially harmful actin filaments from the blood. DBP has also been found to associate with membrane-bound immunoglobulin on the surface of B lymphocytes (15) and with the IgG F, receptor on the membranes of a subpopulation of T cells (16). There has been extensive screening of the human DBP/G, polymorphisms and no G,O homozygotes have been detected (17). This suggests that some or all of DBP’s functions are critical for survival. Despite the structural similarities of albumin, a-fetoprotein, and vitamin D-binding protein, the native proteins are not immunologically cross-reactive. However, antisera to reduced and carboxymethylated RSA or rat cr-fetoprotein are cross-reactive with all three proteins (18). The cross-reactive antibodies appear to recognize sequential determinants in the primary structure that are not exposed during immunization with the native protein. RDBP showed weaker immunological cross-reactivity than RSA or rat a-fetoprotein. In the present studies immunoscreening of cDNA libraries suggested that RLSO is antigenically related to these gene family-related proteins. Given the functional diversity within this family, it was conceivable that sulfite oxidase could be related to them in some way. In order to address this point, we used Western blot and dot blot analyses to examine the cross-reaction of RLSO antibodies to RSA and RDBP and to characterize the determinants involved. MATERIALS
AND
METHODS
Materials. Electrophoresis materials, nitrocellulose, and Affi-Gel15 were purchased from Bio-Rad. “‘I-protein A (>30 or >70 pCi/ng) was from ICN. Freund’s adjuvant was from Difco. Rabbit skeletal muscle was from Pel-Freeze. Rabbit anti-rat albumin antibody was purchased from Cooper Labs. Rat albumin was purchased from Sigma. The material contained a single band as judged by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Protein A-Sepharose 4B and all other biochemicals were purchased from Sigma. Human vitamin Dbinding protein was kindly provided by Dr. John Haddad (Univ. of Pennsylvania). Sulfite oxidasepur$ication. Rat and human sulfite oxidase proteins were purified according to the procedure of Johnson and Rajagopalan (19). To ensure that RSA and RDBP did not contaminate the rat preparation, the rat livers were perfused with ice cold 0.25 M sucrose prior to homogenization to remove as much blood as possible. Purification steps were monitored for RSA with RSA antibody. DEAE-cellulose fractions with the best A,JAzao ratios were pooled, concentrated by Amicon filtration with a PM10 membrane, and fractionated by FPLC (Pharmacia) on a preparative Superose-12 column run in 0.05 M TrisHCl, pH 8.0,O.l mM EDTA. Fractions with AdJAm > 0.8 were further
WITH
RELATED
PROTEINS
169
purified by FPLC Mono Q chromatography using a 30-ml gradient of 0.2-0.45 M NaCl. Fractions with A4,,/Aw = 1.0 were isolated. By all criteria established previously, this is pure enzyme. Sulfite oxidase activity was stained in gels according to the procedure of Cohen (20). DEP purification. Rat DBP was purified by actin affinity chromatography (21). Afli-Gel-15 (25 ml) was washed as described and incubated overnight at 4’C with 25 ml G-actin (5 mg/ml) prepared from frozen rabbit skeletal muscle as described by Pardee and Spudich (22). The gel was extensively washed in depolymerizing buffer (0.01 M Tris-HCl, pH 8.0, 0.2 mM CaCl,, 0.5 mM NasATP, 1 mM NaNs, 0.2 mM DTT). The gel was used within 48 h of its preparation. Rat blood was collected from decapitated animals in the presence of 2 mM EDTA. The red cells were removed by centrifugation at 4000g. Serum was stored at -20°C until use. Serum was thawed, then centrifuged to remove particulate matter before application to the actin affinity column. Serum (20-40 ml) was passed through the column twice before washing and eluting as described. The protein from actin affinity chromatography was further purified by FPLC Mono Q chromatography. The sample in 0.01 M Tris-HCl, pH 8.0, was loaded, then washed with 0.05 M Tris-HCl, pH 8.0. DBP was eluted with a 30-ml gradient of O-O.4 M NaCl. The purity was estimated to be >90% by SDS-polyacrylamide electrophoresis (SDSPAGE). SDS-polyacrylamide gel ekctrophoresis and Western blot analysis. SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (23) in a Bio-Rad Protean electrophoresis unit. Transfer of protein bands from SDS-polyacrylamide gels to nitrocellulose by the LKB Novablot semidry blotter was effected according to the manufacturer’s instructions. Transfer to nitrocellulose paper was done at 0.8 mA/cmx for 1.5 h. The paper was washed in TBS (0.05 M Tris-HCl, pH 8.0, 0.15 M NaCl) then incubated in 5% nonfat dry milk in 0.5X TBS for l-3 h. Incubation with the antibody was carried out in a Seal-AMeal bag in the presence of the blocking agent (2% BSA or milk) at 4°C overnight. A 5% milk solution contains approximately 0.1 mg/ml BSA (24). One-hundred micrograms of IgG was typically used per blot. The blot was washed three times with TBS for 15 min each. The second wash contained 0.1% TX-100. The blot was incubated in a bag with 1.0 pCi ‘?-protein A in TBS with the blocking agent for 3-6 h. The blot was washed as described above and then autoradiographed on Kodak X-OMAT AR film. Dot blots were carried out by spotting protein solution on a nitrocellulose filter. The paper was allowed to dry. All further incubations were as described for Western blots. Immunization protocol. A New Zealand white rabbit was injected subcutaneously at multiple sites with 400 pg RLSO in Freund’s complete adjuvant. One month later a booster injection of 800 pg was given in incomplete adjuvant. The rabbit was bled 1 week later. Antibody to SDS-denatured homogeneous RLSO was prepared by the following method. Approximately 200 pg of protein was subjected to electrophoresis in 7.5% SDS-polyacrylamide gel. The gel was briefly stained with Coomassie blue R-250 so that the location of the RLSO could be identified, then destained. The gel was soaked in water, then the protein bands were cut out with a razor blade. The segment was mashed, then homogenized with 1.5 ml of water and 1.5 ml of complete adjuvant. The emulsion was injected subcutaneously at multiple sites in a New Zealand white rabbit. The polyacrylamide also acts as an adjuvant in this case (25). One month later, a booster injection of 160 pg of the antigen in incomplete adjuvant was given. The rabbit was bled from the ear 7 days after the booster injection. ZgG preparation. Blood was allowed to clot at 4“C overnight. The clot was centrifuged at 4000 rpm in a SS34 rotor for 15 min. The serum was removed and stored at -20°C until needed. Protein A-Sepharose (5 ml) was equilibrated with 50 mM Tris-HCl, pH 8.0,0.15 M NaCl, 0.1 mM EDTA. One milliliter of rabbit serum was passed over the column and rinsed with 1 column vol of buffer. The unbound material was passed over the column a second time. The column was washed until the A,, of the elute was
