Antigenic determinants of the cytoplasmic domain of band 3 from bovine erythrocyte membrane

ARCHIVESOFBIOCHEMISTRYAND BIOPHYSICS Vol. 274, No. 1, October, pp. 130-137,1989

Antigenic Determinants of the Cytoplasmic Domain of Band 3 from Bovine Erythrocyte Membrane RYUICHI

MORIYAMA, MASAAKI

SAYURI KAWAMATSU, YASUSHI TOMIDA. AND SHIO MAKINO’

KONDO,

Llepartwmzt of Food Science and Technology, SCM of Agriculture, Nagoya University, Nagwa, Chikwa-ku, Aichi 464-01, Japan Received December l&1988, and in revised form April 24,1989

Rabbit antibodies were prepared against the cytoplasmic 38K-Da fragment of bovine band 3 and the immunological cross-reactivity with human, murine, rat, and chicken band 3 was examined. The antibodies cross-reacted with human and rodent band 3, indicating that there is an antigenic determinant(s) common to primate and nonprimate species. However, the antibodies did not recognize chicken band 3. Antigenic sites on the 38K-Da fragment were determined via amino acid sequence and immunoblotting analyses of proteolytic peptides of the fragment. Positions of antigenic determinants which were assumed to be common to primate and nonprimate species were mapped to the areas of residues 127-160 and of residues 259-304 in the primary structure of human band 3. Another epitope(s), which is absent in human band 3, existed in a region having a bovine-specific amino acid sequence. In addition, comparison of sequence data from different species showed that a proposed hinge region and a tryptophan-rich region on the cytoplasmic domain of band 3 [P. S. Low et aZ. (1984) J. BioZ. Chem 259,13,0’70-13,076; R. R. Kopito and H. F. Lodish (1985) Nature (London) 316,234-2381 are also conserved in the bovine case. 0 1989 Academic Press, Inc.

Band 3 is a major erythrocyte membrane protein with a molecular weight of 100,000 (1). The protein consists of two distinct domains; a membrane-associated domain and a cytoplasmic domain (2,3). The membrane-associated domain, the carboxyl-terminal portion of band 3, mediates the exchange of inorganic anions across the membrane and spans the membrane several times (4-6). The cytoplasmic domain contains the amino-terminal end of band 3. This domain in human and bovine band 3 supplies binding sites for several glycolytic enzymes as well as hemoglobin, although the physiological role is obscure (7-9). The domain also associates with ankyrin, which in turn interacts with spectrin, and thus aids in anchoring the cytoskeleton to 1To whom correspondence should be addressed. 0003-9861189$3.00 Copyright All rights

0 1989 by Academic Press. Inc. of reproduction in any form reserved.

the membrane (10). In addition, the domain possesses binding sites of band 4.1 and band 4.2 (11, 12), and a predominant antigenic site(s) of band 3 exists in this domain (13,14). Among these proteins interacting with the cytoplasmic pole of band 3, it has been demonstrated that glycolytic enzymes and hemoglobin bind at or near the acidic amino terminus of the cytoplasmic domain in an electrostatic fashion (9, 15). There are indications (16,17) that a predominant antigenic site(s) is located in a region sufficiently distant from the amino terminus of band 3, and that the ankyrin site is close to the antigenic site. However, at present, binding sites on band 3 polypeptide for antibodies, ankyrin, and other cytoplasmic proteins have not been identified. In the present study, we attempted to determine an epitope(s) of the cytoplasmic

130

IDENTIFICATION

OF EPITOPES OF BAND 3

domain of band 3, via amino acid sequence and immunoblotting analyses of proteolytic peptides produced from a 38K-Da fragment of bovine band 3. In order to detect an epitope(s) common to species, we used the 38K-Da fragment which lacks an amino-terminal 12K-Da segment containing a predominant bovine specific-region from the entire cytoplasmic domain (18). We show here that at least three antigenic determinants exist in the 38K-Da fragment of bovine band 3, and that two of them are determinants probably common to primate and nonprimate species. MATERIALS

AND METHODS

Materials. Sepharose 6B was a product from Pharmacia. Immun-Blot assay kit was from Bio-Rad. Lysyl endopeptidase and all other reagents were purchased from Wako Chemicals. Human band 3-coupled Sepharose 6B (ca. 0.5 mg of band 3/ml of gel) was prepared according to the method of Cuatrecasas and Anfinsen (19), using human band 3 isolated as described previously (20). Preparation of ghosts and bond 3. Unsealed ghosts were prepared from bovine, human, rat, and murine blood as described previously (18). Chicken unsealed ghosts were prepared according to the procedure of Jackson (21). Band 3 was preferentially solubilized from the respective unsealed ghosts by 0.15 M NazHPOI-NaHzP04 buffer, pH 7.4, containing 2% nonaethylene glycol n-dodecyl ether (20). After solid sodium dodecyl sulfate (final 3%) was added, the extract was applied to a TSK G30OOSWcolumn (0.75 X 60 cm, Tosoh) which had been equilibrated with 0.1% sodium dodecyl sulfate, 0.2 M NazHPO.,NaHzP04 buffer, pH 7.0, and eluted with the same buffer. The major peak fraction, which contains band 3, was collected and subjected to an immunoblotting assay. Preparation and proteolytic digestion of the cytoplasmic .98K-Dafragment of bovine band 3. The 38KDa fragment of bovine band 3 was prepared as described previously (18). The fragment was treated with lysyl endopeptidase in 10 mM Tris-HCl, 1 mM EDTA, 0.01% NaN3, pH 8.0, at 30°C for 8 h. The final concentration of the enzyme was 50 pg/ml, and hydrolysis was terminated by addition of diisopropylfluorophosphate (0.1 mM final). PurQication of peptides. Peptides produced by lysyl endopeptidase treatment were applied to a Synchropat RP-8 column (250 X 4.1 mm, Synchrom Inc.) which had been equilibrated with 5% acetonitrile, 10 mM NazHP04-NaH,POI buffer, pH 7.0. After the eolumn was washed with the same buffer, peptides were eluted with a linear gradient of acetonitrile (5-40%)

131

over 40 min. The flow rate was 1 ml/min, and the eluate was monitored by measuring the absorption at 220 nm. Fractions A-J in Fig. 2B were collected. Each fraction was rechromatographed on a Synchropac RP-8 column with a linear gradient of acetonitrile (560%) in 10 mM ammonium acetate, pH 7.0. Fractions A, C, and D were separated into two components, respectively, by this buffer system. Peptides denoted as A,,, Abr B, C., Ct,, D,, Dh, E, F, G, H, I, and J (Fig. 2B) were subjected to sequence analysis. Purified peptide I was further digested with lysyl endopeptidase of high concentration (150 rg/ml) at 30°C for 22 h. After the enzyme was inhibited, the products were applied to a TSK G3OOOSWcolumn (0.75 x 60 cm, Tosoh) which had been equilibrated with 0.1% sodium dodecyl sulfate, 0.2 M NazHP04NaH,P04 buffer, pH 7.0, and eluted with the same buffer. The major peak fraction, which contains both the undigested peptide I and its large subfragment referred to as peptide I-l, was collected. Then peptide I-l was isolated on a Synchropac RP-8 column with a linear gradient of acetonitrile (560%) in 10 mM ammonium acetate, pH 7.0. Antibodies. Antiserum against the 38K-Da fragment of bovine band 3 was raised in a young male white rabbit as described previously (18). After ammonium sulfate fractionation, antibodies were purified by DE-52 (Whatman) ion-exchange chromatography. Two types of antibodies differing in cross-reactivity with human band 3 were obtained by the following procedure. Antibodies (3 ml solution containing 10 mg protein) were applied to a human band 3-bound Sepharose 6B column (0.8 X 5 cm) which had been equilibrated with 5 mM NasHPOd-NaHzP04 buffer, pH 7.6, containing 0.15 M NaCl. The column was washed with the same buffer containing 1 M NaCl and then eluted with 50 mM sodium citrate, pH 3.0. The eluate was immediately adjusted to pH 7.6 with 5 M Tris, and dialyzed against 0.5 M NaCl, 20 mM TrisHCl, 0.01% thimerosal, pH 7.6. We refer to the antibody fraction which was adsorbed on the column as (+)hb3-IgG and the unadsorbed fraction as (-)hb&IgG. Analytical procedures. Proteins were analyzed by 5% polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate, using a 0.1 M NazHPO,-NaHzPO, buffer, pH 7.0, as the electrophoresis buffer (18). For peptide analysis, 0.1% sodium dodecyl sulfate/8 M urea-13% polyacrylamide gel electrophoresis was performed according to the method of Kawano and Hamasaki (22). Immunoblotting was carried out by the method of Towbin et aL (23). Sequencing of the peptides was carried out on a JEOL JAS-47K sequence analyzer. Analytical conditions were the same as those described previously (9). RESULTS

Immunweaetivity of rabbit antibodies raised against the 38K-Da fragment of bo-

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TD-

1

2

3

4

5

1

2

3

4

5

FIG. 1. Immunoblotting of bovine, human, murine, rat, and chicken band 3 with (+)hb3-IgG and (-)hb3-IgG. Ten microliters of partially purified band 3 prepared as described under Materials and Methods was electrophoresed on a 5% polyacrylamide slab gel (0.1 X 6.5 X 8.0 cm) containing 0.1% sodium dodecyl sulfate. Then proteins were transfered to a nitrocellulose paper and blotted with (+)hb3-IgG (A) and (-)hb&IgG (B). Lane 1, bovine band 3,0.3 pg; lane 2, human band 3,1.5 pg; lane 3, murine band 3,0.7 pgg;lane 4, rat band 3,0.7 pgg;lane 5, chicken band 3,3 c(g.Preimmune serum did not immunoblot band 3 from any of the species examined here. TD, migration position of the tracking dye.

vine band 3 with human, murine, rat, and chiden band 3. Preliminary experimentation showed that human band 3 is immunoblotted with anti-38K-Da fragment antibodies. Furthermore, when the antibodies were incubated with alkali-stripped human ghosts and followed by centrifugation, the supernatant did not cross-react with human band 3 any more, while conserving the cross-reactivity with bovine band 3. These observations suggest that the cytoplasmic 38K-Da fragment of bovine band 3 possesses at least two antigenie determinants; one is common to both human and bovine band 3 and the other is absent in human band 3. To further characterize the determinants, immunoglobulin fractions, (+)hb&IgG and (-)hb3-IgG, were obtained by means of human band 3coupled Sepharose 6B affinity chromatography, and their cross-reactivity with band 3 from different species was examined. The results are shown in Fig. 1. As expected, (-)hb3-IgG did not recognize human band 3. On the other hand, rat and

murine band 3 cross-reacted with both immunoglobulin fractions. It must be emphasized, however, that the amounts of band 3 required for the appearance of the observable blotting band in these experiments differed among the four species: about five times as much human band 3 was needed as bovine band 3 and about twice as much murine and rat band 3 were needed. Although large excess amounts of chicken band 3 were used, antibodies obtained here did not show any detectable immunoblotting band with chicken band 3 (Fig. 1). Mapping of antigenic peptides on the primary structure of band 3. In order to map the loci of epitopes of the 38K-Da fragment, we attempted to obtain peptides with antigenicity from hydrolyzed products of the fragment. The fragment was cleaved by trypsin, chymotrypsin, and lysyl endopeptidase, and it was observed that degradation products by lysyl endopeptidase exhibit a marked cross-reactivity with anti-38K-Da fragment antibodies.

IDENTIFICATION

A

133

OF EPITOPES OF BAND 3

B

36KLEP12.5K6.5K-

4ab

3.5KTD1

2

0

10 20 30 Retention Time (mid

40

FIG. 2. Fractionation of peptides produced by lysyl endopeptidase treatment of the 38K-Da fragment. (A) The 38K-Da fragment was incubated at 30°C for 8 h in 10 mM Tris-HCl, 1 mM EDTA, 0.01% NaNs buffer, pH 8.0, containing 50 pg/ml of lysyl endopeptidase. After the enzyme was inhibited, electrophoresis was carried out on a 13% polyacrylamide slab gel (0.1 X 6.5 X 11.5cm) in the presence of 0.1% sodium dodecyl sulfate and 8 M urea. Proteins were silver-stained. Lane 1, the 38K-Da fragment (2 pg protein); lane 2, lysyl endopeptidase-treated 38K-Da fragment (1’7pg protein). Arrows a, b, and c show positions of peptides which cross-reacted with anti-38K-Da fragment antibodies. Migration positions of the 38K-Da fragment and lysyl endopeptidase are indicated as 38K and LEP, respectively. Migration positions of the standard proteins used (cytochrome c (&f, 12,500),aprotinin (iW, 6500), and insulin B chain (ilf, 3500)) are also indicated. Molecular weights of peptides obtained from sequence data were not always coincident with those estimated from gel electrophoresis using standard proteins. (B) Proteolytic products of the 38K-Da fragment (lane 2 of A) were applied to a column of Synchropac RP-8 which had been equilibrated with 5% acetonitrile, 10 mM N%HPO,NaH2POI buffer, pH 7.0. After the column was washed with the same buffer, peptides were eluted with a 5-40% linear gradient of acetonitrile. Ultraviolet-absorbing materials were monitored at 220 nm (solid line). The concentration of acetonitrile is also indicated as a broken line. Fractions A-J were collected.

The gel electrophoretic profile in Fig. 2A shows that the 38K-Da fragment was cleaved into several peptides of molecular weight less than 10,000 by lysyl endopeptidase. Three components shown by arrows in Fig. 2A were immunoblotted with anti38K-Da fragment antibodies. Figure 2B shows a chromatographic profile of lysyl endopeptidase-treated 38K-Da fragment. Major peptides A-J were collected, examined for antigenicity, and sequenced. Among these peptides, H and I cross-reacted with antibodies and gave the same electrophoretic mobility as the antigenic components c and a, respectively, shown in Fig. 2A. In the fraction obtained as peptide H, a 5.5K-Da component was detected as shown

by an immunoblotting assay (Fig. 3A). This contaminant, probably peptide I judging from its electrophoretic mobility, had no effect on amino acid sequence analysis. From a comparison of the amino acid sequence of peptide H with that of human band 3 (24), peptide H was assigned to a position between residues 127 and 160 of human band 3 (74% homology), as shown in Fig. 4. Peptide H cross-reacted only with (+)hb3-IgG. Peptide I was immunoblotted with both (+)hb3-IgG and (-)hb3-IgG (Fig. 3B). This peptide consisted of 61 amino acid residues. Forty-seven amino acids starting at a residue 15 (Pro) of this peptide showed 79% homology with residues 259 to 304 of human band 3 (24). However, the sequence

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ET AL.

A

I

12.5 K6.5 K3.5 KTD-

12

3

123

12

3

FIG. 3. Immunoblotting of antigenie peptides with (+)hb3-IgG and (-)bb3-IgG. (A) Peptide H, (B) peptide I; (C) peptide I-l. Two micrograms of each peptide was electrophoresed as described in Fig. 2A using slab gels (0.1 X 6.5 X 8.0 cm) and immunoblotted according to the same procedure as that in Fig. 1. Lane 1, silver stain; lane 2, immunoblot with (+)hb3-IgG; lane 3, immunoblot with (-)hb3IgG. Migration positions of cytochrome c, aprotinin, and insulin B chain are indicated.

Chicken nurinc Human BLl”iW

Chicken Murine HUM!l Bovine

Chicken Hurlne “man Bovine

$kmbrane-associated

domain

FIG. 4. Amino acid sequence of the cytoplasmic domain of bovine, human (24), murine (26), and chicken band 3 (27). Bovine band 3 is aligned to provide optimal homology with others. Residue numbers are given for human band 3. Boxes indicate amino acid identity among the species. The carboxyl-terminal sequence of peptides Cband Db was not identified. Unsequenced regions are shown as broken lines in parentheses and unidentified residues in sequenced portions are represented by the letter x. Amino-terminal amino acids of the 45K-, 39K-, and 38K-Da cytoplasmic fragments and of the membrane-associated domain are indicated by arrows.

IDENTIFICATION

OF EPITOPES

OF BAND

3

135

pole of bovine band 3 is negative (18). In those experiments, Ouchterlony double diffusion analysis was adopted to assay cross-reactivity. By using Western blotting, we showed here that the cytoplasmic domains of band 3 from both primate and nonprimate species share common antigenie determinants. It must be noted, however, that two- to fivefold amounts of human, rat, and murine band 3 were required to give an immunoblotting intensity equivalent to that of bovine band 3. This probably explains why cross-reactivity could not be detected by the less sensitive Ouchterlony double diffusion analysis adopted in previous studies. The weak cross-reactivity may imply that only a part of several sets of antigenic epitopes is shared among primate and nonprimate species, or that the amino acid sequence of common epitope differs slightly among species. On the other hand, we observed here that antibodies against the bovine 38K-Da fragment do not recognize chicken band 3. This is in agreement with a similar result of Jay who showed a lack of cross-reactivity of a rabbit anti-human band 3 antiserum with chicken band 3 (28). In the procedure employed here to determine antigenic sites, some of the epitopes might be destroyed by a proteolytic cleavage, and especially, the procedure might fail to detect conformational epitopes which are made up of noncontiguous amino acid residues. Furthermore, the possibility cannot be ruled out that an antigenic determinant(s) is present in peptides not isolated here. Thus, it would be relevant to point out that there might be an epitope(s) other than those determined here. We were able to map antigenic peptides obtained here on the primary structure of band 3 based on the sequence homology among species. Peptide H was positioned DISCUSSION to residues 127-160 of human band 3. It has been reported that rabbit antise- The peptide contained an epitope(s) for rum directed to the cytoplasmic pole of hu- (+)hb&IgG. Peptide I possessed epitopes for (+)hb3-IgG and (-)hb&IgG. Although man band 3 gives no immunoprecipitate with band 3 from several nonprimate spe- this peptide was mapped to the region of cies (14). We also showed previously that positions 249 to 304 of human band 3, there is no sequence in other species correspondthe cross-reactivity of human band 3 with ing to the amino-terminal 14 amino acid rabbit antiserum against the cytoplasmic

corresponding to the amino-terminal 14 residues of peptide I was not detected in band 3 from other species. Residue 8 of peptide I was lysine, which shows resistance to lysyl endopeptidase attack. However, the Lys-Pro bond was split with a yield of ca. 50% under extreme conditions, i.e., digestion with 150 pg/ml of lysyl endopeptidase at 30°C for 22 h. Purified peptide I-l, a large subfragment of peptide I, gave the following amino - terminal sequence, Pro - Glu - Gly - Ser - Glu - Glu-, clearly indicating that peptide I-l lacks the first 8 residues of peptide I. This peptide showed an electrophoretic mobility corresponding to that of the antigenic component b shown in Fig. 2A. Peptide I-l did not cross-react with (-)hb3-IgG any more, while conserving the cross-reactivity with (+)hb3-IgG, as shown in Fig. 3C. Amino acid sequence of other peptides. In addition to the antigenic peptides H and I, 11 peptides without antigenicity were sequenced. Among these peptides, B and D, were products of a cleavage at a residue other than lysine. The reason for this is not clear. It was shown by sequence analysis that peptides Cb and Db are probably the same peptide but the portion containing the carboxyl-terminus of this peptide could not be determined. Figure 4 shows amino acid alignments of peptides sequenced here, together with the aminoterminal sequences of the 45K-, 39K-, and 38K-Da cytoplasmic fragments of bovine band 3 reported previously (9). In the figure, the amino-terminal sequence of the membrane-associated domain, which was isolated as described previously (25), is also shown. Respective peptides were aligned to provide optimal homology among the sequences of human, murine, and chicken band 3 (24,26,27).

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alignment of peptide I. Furthermore, pep- was also confirmed in the bovine case. Moreover, peptide H (residues 127-160) actide I-l cross-reacted only with (+)hb3IgG. ‘Taken together, it appears that an tually contained an antibody binding epitope(s) for (+)hb3-IgG on peptide I lies site(s) which might be present in human within a region between residue 259 and band 3. Thus, the present results support residue 304 of human band 3. Both regions the proposal of Demuth et al. for the locus of residues 12’7-160and residues 259-304 of of the ankyrin-binding site. The present sequence data only covered bovine band 3 show a fairly high homology with human and murine band 3 (>64%), two-thirds of the entire sequence of the 38K-Da fragment. However, comparison suggesting that the antigenic determinants for (+)hb3-IgG are probably com- of the present results with those of differmon to primate and nonprimate species. ent species shows that some features of the The presence of the common antigenic de- cytoplasmic domain are conserved among terminants in the positions is not incom- species. A proline-rich region, which was proposed by Low et al. as a regulated hinge patible with the previous observations, (17), is fully conserved among human, muthat there are epitopes in an amino-terminal 24K-Da peptide and a remaining 12K- rine, and bovine band 3 at positions 147, Da peptide, respectively, derived from an 175,187, and 190. Also, a region of tryptoamino-terminal 36K-Da piece of human phanyl cluster is highly conserved (residues 75-105). band 3 (13,14). As already noted, the amino-terminal portion of peptide I supplies a binding ACKNOWLEDGMENTS site(s) for (-)hb3-IgG and the first 14 amino acid sequence has no counterpart in We thank Dr. Takuji Sasaki for his help in perother species. This suggests that the 14 forming the sequence analysis. This work was supamino acid sequence forms a bovine-spe- ported in part by a grant awarded to S.M. from Shinsei Shigen Kyokai. cific epitope(s). Nevertheless, (-)hb3-IgG recognized rat and murine band 3. This fact may imply that an epitope(s) for REFERENCES (-)hb3-IgG common to bovine and rodent band 3 is present in a region(s) not identi1. STECK, T. L. (1978) J. Suprawwl Struct. 8,311-324. fied here. 2. STECK, T. L., KOZIARZ, J. J., SINGH, M. K., REDDY, D., AND KOHLER, H. (1978) Biochemistry 17, Among binding sites for cytoskeletal 1216-1222. and cytoplasmic proteins on the cytoplasmic pole of band 3, the ankyrin, band 4.1, 3. APPEL, K. C., AND LOW, P. S. (1981) J. Biol Chem 256,11,104-11,111. and band 4.2 sites are still unidentified. 4. CABANTCHIK, Z. I., KNAUF, P. A., AND ROTHSTEIN, With respect to the ankyrin site, it has A. (1978) B&him Biophys. Ada 515,239-302. been suggested that the site is located near 5. JENNINGS, M.L. (1984)6. Membr. Biol80,105-117. an antibody-binding site in the human 6. JAY, D., AND CANTLEY, L. (1986) Annu. Rev. Biocase (17), and that the site is eonserved them 55,511-538. among different species and, probably, 7. Yu, J., AND STECK, T. L. (1975) J. Biol Chm 250, among different nonerythroid ankyrin9176-9184. binding proteins (26). Demuth et al. 8. SHAKLAI, N., YGUERABIDE, J., AND RANNEY, H. M. (1977) Biochemistry 16.5593-5597. pointed out the presence of a striking con9. MORIYAMA, R., AND MAKINO, S. (1987) Arch. Bit+ served region in the cytoplasmic domain of them. Biophys. 256,606-617. human and murine band 3 and nonery10. HARGREAVES, W. R., GIEDD, K. N., VERKLEIJ, A., throid band 3-like protein (residues 143AND BRANTON, D. (1980) J. Bid Chem 255, 163 in human band 3) (29). They suggested 11,965-11,972. that this region defines a functional site, 11. PASTERNACK, G. R., ANDERSON, R. A., LETO. T.L., probably the ankyrin site, which is conAND MARCHESI, V. T. (1985) J. Bid C~QWL 260, served in both erythroid and nonerythroid 3676-3683. forms of band 3. The high conservation of 12. KORSGREN, C., AND COHEN, C. M. (1988) J. Biol Chem. 263,10,212-10,218. this region, especially residues 153-163,

IDENTIFICATION

OF EPITOPES

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Acta 943.493-500. 26. KOPITO, R. R., AND LODISH, H. F. (1985) Nature &ondm) 316,234-238. 27. Cox, J. V., AND LAZARIDES, E. (1988) Mol. Cell

BioL 8,1327-1335. 28. JAY, D. G. (1983) J. BioL Chew. 258,9431-9436. 29. DEMUTH, D. R., SHOWE, L. C., BALLANTINE, M., PALUMBO, A., FRASER, P. J., CIOE, L., ROVERA, G., AND CURTIS, P. J. (1986) EMBOJ. 5,12051214.