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Biochimica etBiophysicaActa, 405 ( 1 9 7 5 ) 5 1 7 - - 5 2 1 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA Report BBA 3 1 1 9 0

COVALENT STRUCTURE OF FIBRINOPEPTIDES FROM BUFFALOES BREEDING IN ITALY

C. B A L E S T R I E R I , G. C O L O N N A and G. I R A C E

Institute of Biological Chemistry, 1 Faculty of Medicine, University of Naples, Naples (Italy) ( R e c e i v e d J u n e 18th, 1975)

Summary The primary structure of fibrinopeptides A and B from buffaloes breeding ing in Italy has been determined with a view to establishing whether this animal is an autochthonous species or is the result of recent radiation mutation. Some differences exist at the morphologic and physiologic levels between the Indian buffalo and that breeding in Italy, but they do not allow a clear evolutionary line to be traced between these species. The amino acid sequences, if compared with homologous sequences of the Indian buffalo, show one difference in the most variable region of fibrinopeptides A, in particular the substitution of a serine residue by a glycine residue in position 8. This difference supports the hypothesis of the autochthonous origin of the Italian buffalo.

The origin of the domestic buffaloes breeding in Italy is uncertain. Several hypotheses have been made to establish the origin of this species. CampanileCastaldo [ 1 ] supports the idea that the buffalo was introduced in a relatively recent period following the migrations of Mongolian populations coming from Asia towards Europe. Fossil findings from the high Pleistocene, found in the Lazio and in the isle of Pianosa in the Tuscan Archipelago, support the hypothesis of an autochthonous origin of the buffaloes breeding in Italy such as the last type of Bubalus pallesii, Baer, spread at that time throughout the whole of the European area [2]. At present, the Italian buffalo is breeding almost exclusively in Campania and in the lower part of the Lazio region [3]. Actually there exist some differences on the morphologic and physiologic level between the Indian buffalo and that breeding in Italy, but they do not Address a n y c o r r e s p o n d e n c e to: Dr Ciro Balestrieri, Ist~tuto di Chimica Biologica, I FacoltA di

Medicina e Chirurgla, Universit~ degli Studi, Via Costantinopoli 16, 80138 Napoli, Italy.

518

allow a clear evolutionary line to be traced between these species [3, 4]. To elucidate whether the Italian water buffalo is an autochthonous species or is the result of a recent radiation mutation, we have studied the covalent structure of the fibrinopeptides A and B. Fibrinopeptides A and B are very convenient protein fragments for phylogenetical studies involving very close species since they exhibit the highest rate of changes so far studied [5]. These peptides, both composed of about twenty amino acids, are released from fibrinogen by proteolytic action of thrombin in the conversion of fibrinogen to fibrin during blood coagulation. No definite structure is required to display their main function which is to prevent, with their negative charges, the fibirnogen molecules from approaching each other so as to form the fibrin network structure. The sequences of these peptides from buffaloes breeding in Italy, if compared with those reported by Mross and Doolittle [6] for Indian buffalo, show one difference in the most variable region of the fibrinopeptide A. Fibrinogen was prepared from buffalo plasma by the m e t h o d of Blomb~ick and Blomb~ick [7]. The blood was obtained from a single male water buffalo bred in J e m m a Farm (Caserta, Italy). The fibrinogen was incubated with bovine thrombin (EC 3.4.4.13)(350--400 N.I.H. (Sigma) units per mg of protein) at a ratio of 1 mg of thrombin per 1 g of fibrinogen for 9 h at room temperature; the resulting fibrinopeptides A and B were fractionated by Dowex 50-ion exchange column chromatography, as described by Blombiick et al. [8]. Further purification was achieved by high voltage paper electrophoresis at pH 6.5. Both fibrinopeptides A and B were subjected to acid hydrolysis and the amino acid compositions were determined on a Beckman Model 120 C Amino Acid Analyzer [9]. The estimated amino acid content of the fibrinopeptide A was: Asp(2), Ser(2), Glu(3), Gly(5), Ala(2), Val(2), Leu(1), Phe(1), Arg(1). No residue of tryptophan was detected by using the Ehrlich test [10] in the unhydrolyzed peptide. The N-terminal, determined by the Dansyl procedure [ 11 ], was glutamic acid. This composition was n o t consistent with that found by Mross and Doolittle [6] for Indian buffalo fibrinopeptide A. The difference in the amino acid composition was found in the number of serine and glycine residues: in particular, Mross and Doolittle reported the presence of six glycine and one serine residues whereas we found five glycine and two serine residues. To establish the amino acid sequence, a sample of fibrinopeptide A was subjected to chymotryptic hydrolysis for 5 h at 37°C in 0.1 M ammonium bicarbonate. Two peptides were purified by high voltage paper electrophoresis at pH 6.5 and 3.5 and submitted to amino acid composition. Peptide sequences were determined by the Dansyl-Edman procedure as described by Gray and Hartley [12]. Dansyl amino acids were identified by two-dimensional, thinlayer chromatography on polyamide sheets using the solvents reported by Woods and Wang [13]. The results are summarized in Fig.1. The C, peptide showed as an N-terminal glutamic acid, like the whole fibrinopeptide A. It was not necessary to subject the fibrinopeptide A to further hydrolyses since these findings allow the whole sequence to be established unequivocally because of the homology with the artiodactyl fibrinopeptides A. The amino acid analysis of the fibrinopeptide B indicated that the poly-

519 FIBRINOPEPTIDE A CI:

~Glu-Asp-Gly-Ser-Asp-Ala-Val-Ser-Gly-Glu-Phe

C2:

*Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg

FIBRINOPEPTIDE B Tt:

Pyrrolidone carb oxylic acid (Phe,Pro,Thr, Asp,Tyr, Asp,Glu,Gly, Gln,Asp,Asp,Arg,Pro,Lys)

T2!

Leu(Gly,Leu,Gly,Ala)Arg

Fig. 1. Summary o f s e q u e n c e data o b t a i n e d for fibrinopeptides A and B o f Italian w a t e r buffalo. C or T: c h y m o t r y p t i c or tryptic peptides. S e q u e n c e s o f C t and C 2 were d e t e r m i n e d b y the Dansyl-Edman procedure according to Gray and Hartley [12].

peptide contains 21 residues: Asp(4), Thr(1), Glu(3), Pro(2), Gly(3), Ala(1), Leu(2), Tyr(1), Phe(1), Lys(1), Arg(2). Attempts to determine the N-terminal residue failed. This failure is not surprising since the fibrinopeptides B isolated from bovine species show pyrrolidone carboxylic acid as an N-terminal residue [5]. The fibrinopeptide B was then subjected to hydrolysis with trypsin for 4 h at 37°C in 0.1 M ammonium bicarbonate. After tryptic digestion, t w o peptides were isolated by high voltage paper electrophoresis at pH 6.5 and 3.5 and subjected to amino acid composition and end group analysis. The amino acid analysis revealed tha~ the compositions of both tryptic peptides were found to be identical to those of the peptides isolated by Mross and Doolittle [6] from fibrinopeptide B of Indian water buffalo. In addition, one of these peptides was ninhydrin positive and showed leucine as an N-terminal amino acid; the other one, very weakly ninhydrin positive, was stained by a chlorination method [14] and failed to react at the N-terminal. The results are summarized in Fig. 1. Because these results are thoroughly consistent with those found by Mross and Doolittle [6] for the Indian water buffalo fibrinopeptide B, we have n o t insisted on sequencing the tryptic peptide fragments and accept the same arrangement proposed by Mross and Doolittle for the whole fibrinopeptide B. In Table I, the sequences proposed for fibrinopeptides A and B are reported and compared with the homologous sequences of the Indian buffalo. Comparing the sequences, one difference is noted: the presence of a serine

TABLE I COMPARISON OF AMINO ACID SEQUENCES OF FIBRINOPEPTIDES (F) A AND B FROM I TALI AN AND INDIAN WATER BUFFALO PCA: p yrrolidone carboxylic acid Italian FA Indian FA *

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 * *Glu-Asp-Gly-Ser-Asp-Ala-Val-Ser-Gly-Glu-Phe-Leu-Ala-Glu-Gly-Gly.Gly.Val.Arg Glu-Asp-Gly-Ser-Asp-Ala-Val-Gly-Gly-Glu-Phe-Leu-Ala-Glu-Gly.Gly.Gly.Val.Arg 1

2

3

4

5

6

7

S

9

10

11 12

13

14

16

16

17

18 19 20

21

Italian FB

PCA(Phe•Pro•Thr•Asp•Tyr•Asp•G•u•G•y•G•n•Asp°Asp•Arg•Pr••Lys)Leu(G•y•Leu•G•y•A•a)Arg

Indian FA*

PCA(Phe,Pro,Thr)(Asp ,Tyr, Asp,Glu,G|y ,Gin,Asp,Asp ,Arg,Pro, Lys) (Leu,Gly,Leu, Gly,Ala)Arg

*From Mross and Doolitle [6]. **Sequences d e t e r m i n e d by Dansyl-Edman p r o c e d u r e according t o Gray and Hartley [ 12].

520 residue in place of a glycine residue in position 8 of fibrinopeptide A. The paleontological data on the buffalo species does n o t allow taxonomic and phylogenetic relationships to be traced in detail within these species [2, 4]. Buffaloes have been grouped only in a few species, since crossbreeds have produced a series of varieties, more or less fixed in relation to particular environmental conditions, that, with a detailed examination, cannot be considered as distinct species. Both the Italian and Indian buffaloes have always been considered members of the same species (Bos bubalus L.). In this respect the difference found in the sequence of the fibrinopeptides is remarkable. On the other hand, there are animals morphologically very similar (for example the pairs: horse-donkey, dog-fox, zebra I-zebra II) that show differences in their fibrinopeptide sequences [ 5]. The comparative analysis of the data available for fibrinopeptide A sequences shows that two regions can be distinguished in this peptide: the carboxyl-terminal region, that is more constant, and the amino-terminal region, that is less conserved. The highest frequency of changes is observed near position 8 where our difference is found. No large variation of the conformational and functional properties of the fibrinogen molecule can be expected from the substitution of a giycine with a serine residue, the two amino acids having the same value of hydrophobicity [15]. It is w o r t h y of note that this change can result from the substitution of a single base of genetic material (GGU or GGC for glycine -* AGU or AGC for serine). Cape buffalo Italian Indian / water buffalo water buffalo ] \\\

Bovine

Europ/eanbison /

Fig. 2. Particulars o f t h e e v o l u t i o n a r y tree o f f i b r i n o p e p t i d e s A a n d B [5]. Italian w a t e r b u f f a l o is repr e s e n t e d b y a d a s h e d line. T h e b r a n c h l e n g t h s are n o t p r o p o r t i o n a l t o the n u m b e r o f a m i n o acid replacements.

In order to return to the phylogenesis of this animal, our data support the autochthonous hypothesis of the origin of Italian buffalo. We would propose then the scheme shown in Fig. 2 as a modified particular of the larger phylogenetic tree reported by D a y h o f f [5]. This work has been supported by a grant from the Consigiia Nazionale delle Ricerche, Rome, Italy (contratto No. CT 74.00494.06).

521

References 1 2 3 4 6 6 7 8 9 10 11 12 13 14 15

Campanile-Castaldo, M. (1960) Riv. Zootec. 23, 203--206 Maymona, B. (1952) Enc. Agr. It. 1, 1018--1021 Ferrara, B. (1964) Riv. Zootec. 37, 304--315 Pissarewsky, J.Y. (1935) Les buffles, Editions S.N.I.E.-Paris Dayhoff, M.O. (1 972) Atlas of Protein Sequence and Stl~cture, Vol. 5, pp. 47--52 and D-97, N.B.R.F., Washington Mross, G.A. and Doolittle, R.F. (1967) Arch. Biochem. Biophys. 122, 674--684 Blomb~/ck, B. and Blomb~/ck, M. (1956) Arkiv. Kemi 10, 415--418 Blomb~'ck, B., Blomb~'ck, M., Edman, P. and Hessel, B. (1966) Biochim. Biophys. Act~ 115, 371--396 Spaekman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem. 1190--1206 Smith, I. (1963) Nature 171, 43--44 Gray, W.R. (1967) Methods in Enzymology, (C.H.W. Hits, ed.), VoL 11, pp. 139--151, Academic Press, New York Gray, W.R. and Hartley, B.S. (1963) Biochem. J. 69, 379--388 Woods, K.R. and Wang. K.T. (1967) Biochim. Biophys. A c t a 133, 369--370 Rydon, H.N. and Smith, P.W. (1952) Nature 169, 922--923 Nozaky, Y. and Tanford, C. (1971) J. Biol. Chem. 246, 2211--2217