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Biochemical Genetics, Vol. 21, Nos. 7/8, 1983

Hemoglobin Bali (Bovine):/~A18(BI)Lys • His: One of the "Missing Links" Between/~A and ~B of Domestic Cattle Exists in the Bali Cattle (Bovinae, Bos banteng) Takao N a m i k a w a , ~ Osamu Takenaka, 2 and Kenji Takahashi 2 Received 5 Nov. 1982--Final 26 Jan. 1983

The structure of the/3 chain of adult bovine hemoglobin Bali of the Bali cattle was determined and compared to those of t3A, fiB, and other f-chain variants of domestic cattle reported previously, The lysine residue at fA18 was substituted by histidine in t3Balil8. This change requires two base substitutions at the codon and is also found in /3B18. The fib chain differs from the fA chain at residue Nos. 15, 18, and 119. It was concluded that a common ancestor of the f " and fBatifirst diverged from the/3 A chain through the Lys ---* His substitution. This fact indicates that the high degree of dimorphism of the flA and {38 chains in Indian humped cattle is a result of its hybrid origin. An evolutionary tree for the bovine hemoglobin f-chain variants was constructed based on parsimonious evolution and homology with related species. KEY WORDS: biochemical polymorphism; tryptic peptide separation; hemoglobin primary structure; hemoglobin t3-chain evolution; bovine hemoglobin.

INTRODUCTION The f chains of bovine hemoglobins A and B are controlled by two allelic genes. These alleles appear in highly dimorphic states in numerous popula-

This work was supported in part by Grants-in-Aid for Overseas Scientific Survey of Ministry of Education, Science and Culture, Japan (304115 and 404315). Laboratory of Animal Genetics, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464, Japan. 2 Department of Biochemistry, Primate Research Institute, Kyoto University, Inuyama, Aichi 484, Japan. 787 0006-2928/83/0800-0787503.00/0 © 1983PlenumPublishingCorporation

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tions of domestic cattle, especially in the humped cattle (or zebu cattle) (Bangham, 1957; Shreflter and Salisbury, 1959; Crocket et al., 1963; Naik et al., 1969; Singh and Khanna, 1973; Braend, 1972; Osterhoff, 1975; Namikawa, 1980, 1981). The fg chain differs from the fir chain by at least three amino acid residues at Nos. 15, 18, and 119 (Schroeder et al., 1967, 1972). The substitution of Lys (fA) by His (fa) at No. 18 requires two base changes at the codon so that at least four steps are necessary for the differentiation of~ the two chains. Therefore, there must be several intermediate f-chain variants. One of the "missing links" may be distinguished from both of the fA and fib chains by electrophoretic methods because the/3 A differs from the/3 s by two additional positive charges at alkaline pH (Braend, 1972). However, there has been no intermediate f-chain variant in domestic cattle. Recently, a bovine B-chain variant showing the intermediate electrophoretic mobility was found in the Bali cattle of Indonesia (Namikawa and Widodo, 1978; Namikawa, 1981). The Bali cattle is a domesticated form of wild bantengs (Bos banteng) (Mason, 1969). This electrophoretic/3 variant (Hbb-X) occurred at an allelic frequency of 0.802 in the Bali cattle (Namikawa, 1981). In the present study, the B-chain variant was termed fB,li since it was the fifth cattle/3 variant characterized at the level of its primary structure. The structural differences between the/3 Bal~chain and the other f-chain variants of domestic cattle were determined to solve the evolutionary path of these/3 chains and to clarify the origin of dimorphism found in the fA and fib of the humped cattle.

MATERIALS AND METHODS Red blood cells were obtained from 20 adult Bali cattle having Hb Bali in the homozygous state. The Hb A sample was taken from one adult animal of the Hereford breed. Globins were prepared from Hb solution by the method of Teale (1959) and were separated into a and/3 chains by column chromatography using CM-cellulose (Whatman CM-32) (Clegg et al., 1966). The f chains were S-carboxymethylated (Crestfield et al., 1963). S-Carboxymethylated/3 chains were digested with trypsin (Worthington, TPCK treated) at an enzyme/substrate ratio of 1/200 (w/w) a t p H 8.0 for 20 hr at 38°C. Tryptic peptides were separated by cation-exchange chromatography using Aminex A-5 (Bio-Rad Laboratories). A portion (500 ul) of each fraction was mixed with 2 ml of 0.4 M borate-KOH buffer (pH 8.5) and was reacted with 150 ul of fluorescamine solution (3 rag/10 ml acetone). The fluorescence was measured at 475 nm with an excitation wavelength of 370 nm. Descending paper chromatography was performed on Whatman 3 MM paper (Waley and Watson, 1953) for further purification of tryptic peptides

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and also for comparison of R f values between the homologous peptides from the/3 Ba~and/3 A. Amino acid analyses were carried out with a JEOL JLC-6AH or a Hitachi 835 amino acid analyzer. Each sample was hydrolyzed in 6 N HC1 at 110°C for 20 hr. No correction was made for losses during acid hydrolysis. Tyrosine and tryptophan in the peptides were detected spectrophotometrically using a Hitachi 557 Spectrophotometer (Edelhoch, 1967). Some of the tryptic peptides were partially sequenced by the manual Edman degradation method (Edman and Henschen, 1975). The tryptic peptide, T-13, could not be obtained by the above methods due to its very low solubility (Schroeder et al., 1967, 1972). S-Carboxymethylated/3 chain was cleaved at the aspartyl(98)-proline(99) bond by treatment with 75% formic acid containing 7 M guanidine hydrochloride (JaureguiAdell and Marti, 1975). The fragment comprising residues from No. 99 to No. 145 (AP-2) was purified by gel filtration on a Sephadex G-50 column. The AP-2 from the/3BaHchain was sequenced until 134Alathrough T-13 with a JEOL JAS-47K sequence analyzer. The peptide of T-15 of both/3 B"liand/3 A chains was purified from tryptic hydrolysates of the respective AP-2s prepared by digestion at an enzyme/substrate ratio of 1/50 (w/w) at pH 8.0 for 4 hr at 38°C. The nomenclature for the tryptic peptides followed previous work (Schroeder et al., 1967, 1972). RESULTS Separation of o~ and/3 Chains The globins prepared from Hb A and Hb Bali were each separated into two major components by CM-32 column chromatography. These two components were identified as the a and/~ chains from their amino acid compositions. The/~Bali chain was eluted slightly faster than the/31 chain from the column. Separation of Tryptic Peptides Figures la and b shows the cation-exchange chromatograms of the tryptic peptides of the S-carboxymethylated/3Aand/3 Balichains, respectively. Most of the peptides were successfully obtained from these fractions and were further purified by the paper chromatography. The peptide, T-3 b, was not found in the tryptic digests of the /3Bal~ chain. T-4 was purified as N- and C-terminal fragments due to unexpected cleavage as described previously (Schroeder et al., 1972). The yield of T-15 was not satisfactory, because it was further cleaved (Schroeder et al., 1972). Then this peptide was obtained again from AP-2(/399-145), as described later. T-13 was not found in the fractions of

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Aminex A-5 column chromatography for either flA or /3B"~i.Among homologous peptides obtained from the two chains, only T-3 ~b of/3 Bali showed a slightly higher Rzvalue than did T-3 ab of/~A on paper chromatography.

Amino Acid Analyses of the Tryptic Peptides Amino acid compositions of the tryptic peptides of the ~A and/~Balichains were compared. A significant difference was found only in the peptide of T-3 ab. The T-3 ~bof/3 Ayielded 0.93 lysine and no histidine, while the T-3 ~bof/3 B"I~had 0.87 histidine and negligible lysine per molecule of peptide. The tryptic digestion of the/3 A chain produced T-3 ", T-3 b, and T-3 ab due to incomplete cleavage at the (T-3a)-(T-3 b) bond, that is, (Val-Lys)-(Val-Asp . . . . . Arg). On the other hand, the tryptic digestion of/~Bali yielded only T-3 ~b. Actually, the yield of Val-Lys peptide which comes from T-6 and T-3 a in the case of ~A was significantly lower in f/Balithan in/~A (Figs. la and b). This fact together with the amino acid composition suggests that lysine at residue No. 18 is substituted by histidine in T-3 "b of the/3 ~l~ chain. The manual sequencing data of T-3 ab of ~Ba~i, in fact, revealed that the amino acid sequence of its N-terminal part was V a l - H i s - V a l - A s p . . . . (Fig. 4).

Acidic Cleavage of the/3 Chains into AP-I (1-98) and AP-2 (99-145) The elution profile of the/~Ba~icleaved by treatment with formic acid is shown in Fig. 2. Peaks I, II, and III were the intact ~ chain, AP-1, and AP-2, respectively. The absorbance at 280 nm of AP-2 was very low because it has only one tyrosine, while AP-1 has one tyrosine and two tryptophan residues per molecule of peptide. There was no significant difference in amino acid composition between AP-2 fragments from flA and those from flBaJi. The peptide, T-15, was obtained from a tryptic digest of AP-2 (Fig. 3). The N-terminal proline at residue No. 99 of the AP-2 does not react with fluorescamine so that the C-terminal fragment of T-12 (99-103) does not appear in Fig. 3.

Sequencing of AP-2 of the ~Bali Chain The AP-2 fragment of/~Baliw a s sequenced from the N terminus (Fig. 4). The sequence of T-13 of ~Bali was identical to that of the /~A chain which was assumed by homology from the amino acid composition of the peptide by Schroeder et al. (1967). The peptide, T-14 b, was also sequenced, since concrete data on the sequence have been lacking (Fig. 4). From the amino acid composition, Schroeder et al. (1967) proposed G l u - P h e - T h r - P r o - A s p - V a l -

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G l n - A l a - L e u - P h e - G l n - L y s for the sequence of T- 14b, and later Schroeder et al. (1972) rectified this to G l u - P h e - T h r - P r o - V a l - L e u - G l n - A l a - A s p - P h e -

Gln-Lys. The present sequence data confirmed that the latter is correct. DISCUSSION Lysine at residue No. 18 is common to different species of Artiodactyla such as yak (Lalthantluanga and Braunitzer, 1981), sheep (Boyer et al., 1967; Wilson et al., 1966, 1970), and goat (Adams et al., 1968; Huisman et al., 1967; Wrightstone et al., 1970). The direction of substitution is, therefore, considered to be from lysine to histidine. This change requires two base substitutions and is a very rare case found in closely related species. It is not likely that this substitution has occurred in respective fib and/~Bali in parallel. This means that a common ancestor of fib and fBa]i first diverged from the/~A and then it evolved to the present two chains. The most likely evolutionary path for the f-chain variants is depicted in Fig. 5. The network linking the variants was assigned on the basis of the most parsimonious evolution among them. The root of the tree was fixed by homologies to related species such as bovine (Lalthantluanga and Braunitzer, 1981), sheep (Boyer et al., 1967; Wilson et al., 1966, 1970), goat (Adams et al., 1967; Huisman et al., 1967; Wrightstone et al., 1970), and pig (Braunitzer and K/Shler, 1966). It was noticed that the fB of domestic cattle showed the closest evolutionary relationship to the fBa~i. The allelic dimorphism with the f a and fib chains is well known in humped cattle (Naik et al., 1969; Singh and Khanna, 1973; Braend, 1972; Osterhoff, 1975; Namikawa, 1980, 1981). The reason for the coexistance of the two variants in a population has often been discussed because they are differentiated by multiple amino acid substitutions. This contrasts with the

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Fig. 5. The most likely evolutionary path of bovine hemoglobin chains. Data on the variants other than ~aal~are cited from the previous report (Schroeder et al., 1972).

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fact that almost all of the human/3 variants can be explained by a single amino acid substitution. There are two possible explanations for the high degree of dimorphism with the/3 A and 13Bchains in humped cattle. One is that the/3 a has evolved from the/3 a in an isolated domestic population and then the new allele has expanded its distribution to other populations. But this assumption cannot explain why the/3 BaHof Bali cattle (Bos banteng) is a transitional form from t3A to /3B. The other explanation is that the common ancestor of/3 a and /3Bal~ differentiated from the/3 A chain in a wild species before the domestication of the humped type of cattle. Although it is not obvious what kinds of species have contributed genetically to an original stock of humped cattle, a wild species carrying the/3 B chain may be more closely related to the Bali cattle or its wild form, bantengs, than to aurochs (Bos p r i m i g e n i u s ) . The auroch is generally accepted to be a major wild ancestor of the domestic cattle. The possibility of hybrid origin of the humped cattle has als0 been discussed based on the several protein polymorphisms which involve multiple amino acid substitutions (Manwell and Baker, 1976). The present study indicated that the/3 aal~ chain is a possible evolutionarily transitional type from the ~a to the/3 a chain and exists in the "different" species. This fact provides evidence supporting the theory of the hybrid origin of humped cattle. ACKNOWLEDGMENTS The authors thank Dr. T. Sasaki, Laboratory of Physical Chemistry, Faculty of Agriculture, Nagoya University, for the guidance in automatic sequencing. They are also indebted to Dr. K. Nozawa, Department of Variation Research, Primate Research Institute, Kyoto University, and Dr. K. Kondo, Laboratory of Animal Genetics, Faculty and Agriculture, Nagoya University, for their discussion and encouragements. REFERENCES

Adams, H. R., Boyd, E. M., Wilson, J. B., Miller, A., and Huisman, T. H. J. (1968). The structure of goat hemoglobins. III. Hemoglobin D, a /3 chain variant with one apparent amino acid substitution (21Asp ~ His). Arch. Biochem. Biophys. 127:398. Bangham, A. D. (1957). Distribution of electrophoreticallydifferent hemoglobinsamong cattIe breeds of Great Britain. Nature Lond. 179:467. Boyer, S. H., Hathaway, P., Pascasio, F., Bordley, J., Orton, C., and Naughton, M. A. (1967). Differences in the amino acid sequences of tryptic peptides from three sheep hemoglobin/3 chains..1. Biol. Chem. 242:2211. Braend, M. (1972). Studies on the relationships betweencattle breeds in Africa, Asia and Europe: Evidence obtained by studies of blood groups and protein polymorphisms.World Rev. Anita. Prod. 8:9. Braunitzer, G., and K~Shler, H. (1966). Zur Phylogenie des H~imoglobinmolek~ils:Untersuchungen am H~moglobindes Schweines. Z. Physiol. Chem. 343:290. Clegg, J. B., Naughton, M. A., and Weatherall, D. J. (1966). Abnormal human hemoglobins: Separation and characterization of the c~ and /3 chains by chromatography, and the

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determination of two new variants, Hb Chesapeake and Hb J(Bangkok). J. Mol. Biol. 19:91. Crestfield, A. M., Moore, S., and Stein, W. H. (1963). The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. Biol. Chem. 238:622. Crocket, J. R., Koger, M., and Chapman, H. L., Jr. (1963). Genetic variations in hemoglobins of beef cattle. J. Anita. Sci. 22:173. Edelhoch, H. (1967). Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948. Edman, P., and Henschen, A. (1975). In Needleman, S. B. (ed.), Protein Sequence Determination, 2nd ed., Springer, New York, pp. 232-279. Huisman, T. H. J., Adams, H. R., Dimmock, M. O., Edwards, W. E., and Wilson, J. B. (1967). The structure of goat hemoglobins. I. Structural studies of the/3 chains of hemoglobins of normal and anemic goats. J. Biol. Chem. 242:2534. Jauregui-Adell, J., and Marti, J. (1975). Acidic cleavage of the aspartyl-proline bond and the limitations of the reaction. Anal. Biochem. 69:468. Lalthantluanga, R., and Braunitzer, G. (1981). The primary structure of the Bl- and flU-chains of yak hemoglobins (Bovidae). Hoppe-Seyler Z. Physiol. Chem. 362:1405. Manwell, C., and Baker, C. M. A. (1976). Protein polymorphisms in domesticated species: Evidence for hybrid origin? In Karlin, S., and Nevo, E. (eds.), Population Genetics and Ecology, Academic Press, New York, pp. 105-139. Mason, I. L. (1969). A World Dictionary o f Livestock Breeds, Types and Varieties, 2nd ed., Common Wealth Agricultural Bureaux, Farnham Royal, Bucks, England. Naik, S. N., Sukumaran, P. K., and Sanghvi, L. D. (1969). Hemoglobin polymorphism in Indian zebu cattle. Heredity Lond. 24:239. Namikawa, T. (1980). Genetical aspects of domestication and phylogeny in the cattle. Jap. J. Zootech. Sci. 51:235. Namikawa, T. (1981). Geographic distribution of bovine hemoglobin-beta (Hbb) alleles and the phylogenetic analysis of the cattle in eastern Asia. Z. Tierzucht. Zuchtbiol. 98:151. Namikawa, T., and Widodo, W. (1978). Electrophoretic variations of hemoglobin and serum albumin in the Indonesian cattle including Bali cattle (Bos banteng). Jap. J. Zootech. Sci. 49:817. Osterhoff, D. R. (1975). Hemoglobin types in African cattle. J. S. Afr. Vet. Assoc. 46:185. Schroeder, W. A., Shelton, J. R., Shelton, J. B., Robberson, B., and Babin, D. R. (1967). A comparison of amino acid sequences in the B-chains of adult bovine hemoglobins A and B. Arch. Biochem. Biophys. 120:124. Schroeder, W. A., Shelton, J. R., Shelton, J. B., Apell, G., Huisman, T. H. J., Smith, L. L., and Carr, W. R. (1972). Amino acid sequences in the B-chains of bovine hemoglobins C-Rhodesia and D-Zambia. Arch. Biochem. Biophys. 152:222. Shrefller, D. C., and Salisbury, G. W. (1959). Distribution and inheritance of hemoglobin variations in American cattle. J. Dairy Sci. 42:1147. Singh, H. P., and Khanna, N. D. (1973). Hemoglobin-C in Kumaoni-Hill cattle. Indian Vet. J. 50:239. Teale, F. W. J. (1959). Cleavage of the haem-protein link by acid methylethylketone. Biochim. Biophys. Acta 35:543. Waley, S. G., and Watson, J. (1953). The action of trypsin on polylysine. Biochem. J. 55:328. Wilson, J. B., Edwards, W. C., MacDaniel, M., Dobbs, M. M., and Huisman, T. H. J. (1966). The structure of sheep hemoglobins. II. The amino acid composition of the tryptic peptides of the non-a chains of hemoglobins A, B, C, and F. Arch. Biochem. Biophys. 115:385. Wilson, J. B., Miller, A., and Huisman, T. H. J. (1970). Production of hemoglobin C in the moufllon (Ovis musimon Pallas, 1811) and the barbary sheep (Ammotragus lervia Pallas, 1977) during experimental anemia: Amino acid composition of tryptic peptides from the BB and Bc chains. Biochem. Genet. 4:677. Wrightstone, R. N., Wilson, J. B., Miller, A., and Huisman, T. H. J. (1970). The structure of goat hemoglobins: IV. A third/3 chain variant (BE) with three apparent amino acid substitutions. Arch. Biochem. Biophys. 138:451.