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J. Biochem. 97, 1487-1492 (1985)

Fibrinopeptides A and B of Japanese (Macaca fuscata) and Patas Monkey ( patas): Their Amino Acid Sequences, Restricted Mutations, and a Molecular Phylogeny for , , and

Shin NAKAMURA, Osamu TAKENAKA, and Kenji TAKAHASHI1

Department of Biochemistry, Research Institute, Kyoto University, Inuyama, Aichi 484

Received for publication, December 10, 1984

Amino acid sequences of fibrinopeptides A and B from the , Macaca fuscata (Japanese monkey) and the , Erythrocebus patas (patas monkey) were estab lished. Fibrinopeptides A of the monkeys had a sequence identical with those of is 10 5 1 baboons: Ala-Asp-Thr-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg. Fibrinopeptides B were 9-residue, "short," peptides with the sequences Asn-Glu 6 3 6 3 Glu-Ser-Leu-Phe-Ser-Gly-Arg for M. fuscata and Asn-Glu-Glu-Val-Leu-Phe-Gly Gly-Arg for E. patas. The sequence of the B peptide of M. fuscata differed from

5 that of a close-related species, M. mulatto (rhesus monkey), at a single site, Leu

5 (M.f)•¨Pro (M.m.). A single replacement between the B peptides of E. patas 6 6 and Cercocebus aethiops (), Val (E.p.)•¨Gly (C.a.), was detected. A

phylogenic relationship of macaques, guenons, and baboons, named

(), was deduced from the sequence data. A selective rather than random amino acid replacement was observed in the B peptides of these Old

World monkeys, suggesting a restricted mutation of their fibrinopeptides during

primate evolution.

The amino acid sequences of fibrinopeptides A the sequences of both peptides were found to be and B from many species of were investi very variable among species; they show the gated to elucidate the gelation mechanism of fi fastest "evolutionary rates" among known peptides brinogen, because thrombin-mediated removal of and proteins (2, 3). Thus, the A and B peptides the A and B peptides is a trigger for fibrinogen should be useful markers to investigate evolution fibrin transformation (1). Through these studies, ary processes and phylogenic relationships of pri mates on a molecular level. For this reason, the sequences of several primate fibrinopeptides A and 1 Present address: Department of Biophysics and Bio B have been determined (4-11). chemistry, Faculty of Science, The University of Tokyo, In a recent study (12), we reported a new Hongo, Bunkyo-ku, Tokyo 113. Abbreviations: N-, amino; TCA, trichloroacetic acid. molecular phylogeny for baboons based on the

Vol. 97, No. 5, 1985 1487 1488 S. NAKAMURA, O. TAKENAKA, and K. TAKAHASHI sequences of their fibrinopeptides B, and uneven Sequence Analysis-Amino acid analysis of evolutionary rates of the peptides. The sequences the peptides was performed with a JEOL 6AH of the fibrinopeptides A and B of Japanese monkey amino acid analyzer after hydrolysis with 6 m HCl (Macaca fuscata) and patas monkey (Erythrocebus at 110•Ž for 24 h. The amino acid sequences of patas) were determined in the current study. The the peptide samples were determined by the man macaque and the guenon in addition to the ual Edman degradation method (14). Phenylthio are typical Old World monkeys, Cercopithecinae. hydantoin-amino acids were identified by the Sequence data of the A and B peptides of ma method of Kulbe (15) and/or by high-performance caques and guenons are essential to elucidate the liquid chromatography under the conditions de evolutionary characteristics of the Cercopithecinae. scribed previously (16, 17). The dansyl method

A phylogenic relationship for the Old World mon (18) was also used for sequencing. keys deduced from the sequence data is also dis cussed. RESULTS

MATERIALS AND METHODS Fibrinopeptide A fractions, Fraction A in Fig. IS,

derived from Japanese monkey (M. fuscata) and Plasma-Japanese monkey (M. fuscata) plas patas monkey (E. patas) fibrinogens yielded two ma was pooled from five individuals of the Taka peptide fractions, A-‡T and A-‡U, upon high-voltage hama troop. Patas monkey (E. patas) plasma was paper electrophoresis at pH 3.5, in common (Fig. obtained from five individuals of different troops. 2S). Peptides A-‡U were major components, and Preparation of Fibrinogen-Fibrinogen was the amino acid compositions (16 residues) and the purified from the pooled plasma by the ethanol amino (N)-terminus, alanine, were identical (Table citrate method (13). The purified fibrinogen gave IS). Peptides A-‡T were minor components with a a clottability of 93% and showed three protein yield of 8-12 % and again the amino acid com bands, Aƒ¿, BƒÀ, and ƒÁ, upon SDS-polyacrylamide positions (15 residues) and N-terminus, aspartic gel electrophoresis of the reduced sample. acid, were identical (Table IS). These results show Isolation of Fibrinopeptides A and B-Twenty that peptide A-‡U is fibrinopeptide A and that five ml of fibrinogen solution (2 mg/ml) in 0.1 M 16 ammonium acetate (pH 6.9) was supplemented peptide A-‡T is the des-Ala A peptide, named with soybean trypsin inhibitor (25 ƒÊg/ml, Sigma) fibrinopeptide Y (5), as described previously (12). and incubated with thrombin (2.5 units/ml, Parke Fibrinopeptide B fractions, Fraction B in Fig. 1S,

Davis) at 37•Ž for 4 h. After removal of the of both monkeys also gave two peptide fractions, fibril clot, the supernatant was treated with ice B-‡T and B-‡U, upon the paper electrophoresis (Fig. cold 5 % trichloroacetic acid (TCA), and insoluble 2S). Each major component, peptide B-‡U, con materials were removed by centrifugation (8,000 sisted of 9 amino acid residues, and their N rpm •~ 20 min). Then TCA was removed by an terminus, asparagine, was identical, although their extraction with ice-cold ethyl ether (five times). amino acid compositions differed in serine, glycine,

The resulting aqueous phase containing fibrino and valine (Table IIS). This indicates that each pep peptides A and B was concentrated by lyophi tide B-‡U is fibrinopeptide B, because all fibrino lization and fractionated by gel filtration on a peptides B of other macaques (11), guenon (4),

Sephadex G-25 column (1.7 •~ 142 cm) equilibrated and baboons (12) possess 9 constituent residues with 0.1 M NH4HCO3 (pH 8.0) (Fig. IS). The and the same N-terminus as peptide B-‡U of M. peptide fractions, Fractions A and B in Fig. IS, fuscata and E. patas. The minor component, pep were each purified by high-voltage paper electro tide B-‡T, of each monkey had identical amino acid phoresis at pH 3.5 (Fig. 2S). Thermolysin frag composition to the corresponding peptide B-‡U, ments of peptide A (A-‡U in Fig. 2S) and ƒ¿-chymo fibrinopeptide B, but differed in the N-terminus, trypsin fragments of peptide B (B-‡U in Fig. 2S) aspartic acid. Thus, peptide B-‡T was presumably were isolated by paper electrophoresis (Fig. 3S). derived from the B peptide by deamination of

The purified peptide fragments, A-‡U-1, A-‡U-2, N-terminal asparagine. This is consistent with the

B-‡U-1, and B-‡U-2, were used for sequence analysis. more cathodic nature of peptide B-‡T than peptide

J. Biochem. MOLECULAR EVOLUTION OF PRIMATE FIBRINOPEPTIDES 1489

Fig. 1. Amino acid sequences of fibrinopeptides A and B of Japanese monkey (M. fuscata) and patas monkey (E. patas). Th, cleavage site by thermolysin. Ch, cleavage site by a-chymotrypsin.

B-‡U upon paper electrophoresis (Fig. 2S). 14 amino acid residues and a masked N-terminus,

Fibrinopeptides A (A-‡U) and B (B-‡U) were pyroglutamic acid. However, 9-residue, "short," each digested with thermolysin and a-chymotryp B peptides with N-terminal asparagine were ob

sin. Thermolysin fragments, A-‡U-1 and A-‡U-2, tained from M. fuscata and E. patas (Table ‡U).

and a-chymotrypsin fragments, B-‡U-1 and B-‡U-2, The short B peptide has also been reported for

were isolated by high-voltage paper electrophoresis baboons (12), other Asian macaques (11), and

(Fig. 3S), and the resulting peptides were subjected another guenon (4), which are typical Old World to sequencing. Figure 1 shows the amino acid monkeys belonging to Cercopithecinae. Thus, the

sequences of the fibrinopeptides A and B of the short peptide is characteristic of fibrinopeptides B

macaque and guenon. The A peptides of M. of the Old World monkeys. fuscata and E. patas had identical sequence (Fig. In primate fibrinopeptides B, positions 2, 3, 1). On the other hand, two amino acid replace 4, 5, 6, and 8 in the 9 amino acid residues from

ments were observed between the B peptides of the the carboxyl-terminus are variable sites. Positions

monkeys, serine (M.f) to glycine (E.p.) and serine 3, 5, and 6 were the only sites of amino acid

(M.f.) to valine (E.p.) at positions 3 and 6, respec replacement of the Cercopithecinae B peptides

tively. This supports the previous observation (Fig. 2). Interestingly, replacement in the B pep that the B peptides have changed more rapidly tides was observed at genus-specific sites, i.e., posi

than the A peptides during primate evolution (12). tion 3 for baboons (Papio), and positions 5 and 6

for macaques (Macaca). The specific sites for the

DISCUSSION ape B peptides were at positions 3 and 5 (12). These results suggest selective rather than random

Primate fibrinopeptides A are relatively conserva amino acid replacement in the B peptide during

tive to amino acid replacement and only a single evolution of these , even though random 14 14 nature has been accepted for mutation of the amino acid change, Thr•¨Ser, has been observed mammalian fibrinopeptides B (19, 20). More among Old World monkeys, apes,2 and human highly selective amino acid replacement has been (12). The absence of any amino acid replacement observed in primate hemoglobins (21, 22). The between the amino acid sequences of the A pep mechanism for the selective mutation of these tides of Japanese monkey (M. fuscata) and patas primate proteins is not clear, but may result from monkey (E. patas) certainly results from conser a restriction upon base substitution of the corre vatism to amino acid change (Fig. 1). Primate sponding genes during primate evolution. This fibrinopeptides B have been characterized as having selective mutation may cause a "slow down" in the

evolutionary rate of the primate proteins. 2 Apes: siamang, gibbon, orang-utan, gorilla, chim Rhesus monkey (M. mulatta) and Japanese panzee. monkey are close-related species that can produce

Vol. 97, No. 5, 1985 1490 S. NAKAMURA, O. TAKENAKA, and K. TAKAHASHI

a hybrid with fertility. The amino acid sequences not support this . The mutation dis of their fibrinopeptides B differed at a single site, tance separating green and patas monkeys can be 5 5 calculated to be 1.0 from the sequence difference Leu (M.f.)•¨Pro (M.m.) (Fig. 2), and hence their "mutation distance (2)" can be estimated to be 1 of their B peptide (Fig. 2). The values of 2.2 and .0. 4.0 have been estimated as maximum inter-species Green monkey (Cercocebus aethiops) and patas and inter-genus distances, respectively (24). Judg monkey (E. patas) were classified into different ing from these values, the distance between green genera in classical taxonomy (23). Our results do and patas monkeys, 1.0, is smaller than the maxi mum inter-species distance and corresponds to that between the close-related macaques, rhesus and Japanese monkeys. Thus, green and patas mon keys are close-related species and can be placed in the same genus, not in separate genera. Figure 3A shows a cladogram representing a phylogenic relationship for macaques, guenons, and baboons deduced from the amino acid se quences of their fibrinopeptide B. Evolutionary processes separating the species are also inferred from the sequence data. In this cladogram, ge lada baboon ( ) was placed in Fig. 2. Amino acid replacements found among fibrino the same position as patas monkey. However, peptides B of Cercopithecinae, macaques, guenons, and on the basis of our immunochemical studies by baboons. Sequence data: M. mulatta (4), M. nemestrina enzyme-linked immunosorbent assay using anti (11), C. aethiops (4), P. anubis (12), P. hamadryas (12), and Th. gelada (12). Amino acid residues are ex Japanese monkey plasma serum, baboons (Th. pressed in single letter codes: E (Glu), F (Phe), G (Gly), gelada, P. anubis, and P. hamadryas) and guenons H (His), L (Leu), N (Asn), P (Pro), R (Arg), S (Set), (C. aethiops and E. patas) could be classified into V (Val). separate groups, i.e. genera (Tsutsumi et al., un

Fig. 3. A molecular phylogeny for macaques, guenons, and baboons, deduced directly from the amino acid sequences of their fibrinopeptide B (A), and the modified phylogeny incorporat ing the results of immunochemical studies (B). The sequence for an ancestral Cercopithecinae 5 1 fibrinopeptide B, N-E-E-G-L-F-G-G-R, could be assumed from the sequence data in Fig. 2 on the "maximum parsimony principle (26)." Positions and residues shown in the phylogeny indicate amino acid replacement from the ancestral B peptide during species differentiation.

J. Biochem. MOLECULAR EVOLUTION OF PRIMATE FIBRINOPEPTIDES 1491 published data). This observation confirmed the 10. Wooding, G.L. & Doolittle, R.F. (1972) J. Human phylogeny concerning baboons and guenons de Evol. 1, 553-563 duced previously by Sarich and Cronin (25). The 11. Nakamura, S., Takenaka, 0., & Takahashi, K. unexpected position of gelada baboon in the (1979) Zool. Magazine (in Japanese) 88, 678 cladogram for the Old World monkeys may reflect 12. Nakamura, S., Takenaka, 0., & Takahashi, K. unusual characteristics of fibrinopeptide B of ge (1983) J. Biochem. 94,1973-1978 13. Doolittle, R.F., Schubert, D., & Schwartz, S.A. lada baboon. The B peptide evolution appeared (1967) Biochim. Biophys. Acta 118, 456-467 to be markedly restricted, i.e. "slowed down," 14. Edman, P. & Henschen, A. (1975) in Protein Se and its evolutionary rate was one-tenth to one quence Determination (Needleman, S.B., ed.) pp. fifth of those of other baboons and macaques (12). 232-279, Springer-Verlag, Berlin Thus, a modified phylogeny for the monkeys can 15. Kulbe, K.D. (1974) Anal. Biochem. 59, 564-574 be deduced from the combination of B peptide 16. Ishikawa, C., Nakamura, S., Watanabe, K., & sequence data and immunochemical studies, as Takahashi, K. (1979) FEBS Lett. 99, 97-100 shown in Fig. 3B. 17. Omichi, K., Nagura, N., & Ikenaka, T. (1980) J. Biochem. 87, 483-489 18. Gray, W.R. (1972) in Methods in Enzymology (Hirs, REFERENCES C.H.W. & Timasheff, S.N., eds.) Vol. 25, pp. 333 1. Doolittle, R.F. (1975) in The Plasma Proteins 344, Academic Press, Inc., New York 19. Blomback, B. & Blomback, M. (1968) in Chemo (Putnam, F.W., ed.) Vol. 2, pp. 109-161, Academic taxonomy and Serotaxonomy (Hawkes, J.G., ed.) Press, Inc., New York 2. Dayhoff, M.O. & Barker, W.C. (1972) in Atlas of pp. 3-20, Academic Press, Inc., London 20. Doolittle, R.F. (1979) in The Proteins (Neurath, H. Protein Sequence and Structure (Dayhoff, M.O., ed.) Vol. 5, pp. 49-52, National Biomedical Re & Hill, R., eds.) Vol. 5, pp. 1-118, Academic Press, Inc., New York search Foundation, Washington, D.C. 3. Wilson, A.C., Carlson, S.S., & White, T.J. (1977) 21. Matsuda, G. (1975) in Molecular Anthropology Annu. Rev. Biochem. 46, 573-639 (Goodman, M. & Tashian, R.E., eds.) pp. 223-237, Plenum Press, New York 4. Blomback, B., Blomback, M., Grondahl, N.J., 22. Maita, T. & Matsuda, G. (1980) in XXXIth Sym Guthrie, C., & Hinton, M. (1965) Acta Chem. Scand. 19, 1788-1789 posium on Protein Structure (in Japanese), pp. 101 104, 5. Blomback, B., Blomback, M., Edman, P., & Hessel, 23. Hill, W.C.O. (1970) in Primates: Comparative B. (1966) Biochim. Biophys. Acta 115, 371-3966 Anatomy and Taxonomy Part VI, pp. 533-551 and . Doolittle, R.F., Glasgow, C., & Mross, G.A. (1969) 694-704, Edinburgh University Press, Edinburgh Biochim. Biophys. Acta 175, 217-219 24. Nakamura, S. (1980) Monkey (in Japanese) 24, 6-13 7. Doolittle, R.F. & Mross, G.A. (1970) Nature 225, 25. Sarich, V.A. & Cronin, J.E. (1976) in Molecular 643-644 Anthropology (Goodman, M. & Tashian, R.E., eds.) 8. Mross, G.A., Doolittle, R.F., & Roberts, B.F. (1970) pp. 141-170, Plenum Press, New York Science 170, 468-470 26. Moore, G.W., Barnabas, J., & Goodman, M. (1973) 9. Doolittle, R.F., Wooding, G.L., & Riley, M. (1971) J. Theor. Biol. 38, 459-485 J. Mol. Evol. 1, 74-83

Vol. 97, No. 5, 1985 1492 S. NAKAMURA, O. TAKENAKA, and K. TAKAHASHI

Supplemental Materials

Table IS. Amino acid compositions and N-terminal residues of fibrinopeptide A and its derivative from M.fuscata and E. ap tas,

1 Calculated from the initial amount of fibrinogen used.

Table 51S. Amino acid compositions and N-terminal residues of

fibrinopeptide a and its derivativefrom M.fuscata and E.patas ,

1Calculated from the initial amount of fibrinogen used.

Fig. 1S. A typical gel filtration pattern of monkey fibrin clot

supernatant. A concentrated clot supernatant obtained from

H. fuscata or E. pat fibrinogen was gel filtered on a Sephadex

C-25 column (1.7 •~ 142 cm) equilibrated with 0.1 M NH4HC03. Fig. 3S. Purification of thermolysin peptides of A-‡U and

ƒ¿ -chymotrypsin peptides of B-‡U Fraction A (crude fibrinopeptide A) and Fraction B (crude . A-‡U, fibrinopeptide A, of each

fibrinopeptide B) were each purified further. monkey was digested with thermolysin (enzyme : peptide , 1 : 10,

by weight) in 0.1 M NH4HC03(pH 8.0) at 40•Ž for 24 h . B-‡U,

Fig. 2S. High-voltage paper electrophoresis of crude fibrinopeptide B, of each monkey was digested with ƒ¿-chymotrypain

fibrinopeptides A and B. Paper electrophoresis was performed (enzyme : peptide, 1 : 10, by weight) in 0.1 M NH4HC0 3 (pH 8.0) at 3,000 V per 57 cm at pH 3.5 in pyridine-acetic acid-water at 37•Ž for 5 h. The digests were subjected to high-voltage

(1:10:289, by vol.), using methyl green (MG) as a tracking dye. paper electrophoresis at pH 3.5 under the same conditions as

After electrophoresis, guide strips 1 cm wide were stained with described in Fig. 2S. The resulting peptides, A-‡U-1 , A-‡U-2,

0.25 % ninhydrin (•›) and with 0.02 % phenanthrane qulnone for B-‡U-1, and B-‡U-2, were used for sequence analysis .

arginine-containing peptides (•œ). Peptides A-‡T, A-‡U, B-‡T and A, Electrophoretogram of thermolysin digest of A-‡U of both monkeys .

B-‡U were eluted with 0.01 H NH4OH and used for sequence or amino B, Electrophoretogram oftƒ¿-chymotrypsin digest of B-‡U of both

.old analysis. monkeys.

J. Biochem.