J./Viol. Evol. 9, 343-347 (1977)

Journal of Molecular Evolution © by Springer-Verlag 1977

Primitive Haemoglobin Brian Tiplady 1 and Morris Goodman 2 1 Astra Clinical Research Unit, 65, Queen Street, Edingburgh EH2 4NA, United Kingdom 2 Department of Anatomy, Wayne State University, Detroit, Michigan 48201, USA

Summary. The sequences of Petromyzon and Aplysia globins are compared with the postulated vertebrate and mollusc-vertebrate ancestors to see if differences exist in the rates of evolution of different types of residue positions. Between the mollusc-vertebrate ancestor and Aplysia globin there is no very striking pattern of changes except that the interior positions are relatively conserved. In the evolution of Petromyzon haemoglobin, the % 32 contact area is relatively conserved. The homopolymeric binding of lamprey Hb seems to be a primitive function. Key words: Evolution -- Haemoglobin - Cooperativity - Lamprey -- Maximum parsimony.

Introduction A genealogy of haemoglobin has been reported, constructed according to the maximum parsimony approach (Goodman et al., 1974; Goodman et al., 1975). The reconstructed ancestral sequences derived from this genealogy were used to compare the rates of mutation of various parts of the haemoglobin molecule at different stages of the evolution of mammalian alpha and beta chains. The variations ~n nucleotide substitution rates were interpreted in terms of Darwinian selection, the emergence of a new function being followed by a rapid rate of evolution, which then slows down once the molecule has been optimised. It seemed that these data could also assist in answering a different kind of question, that of whether the globins of "lower" animals represent primitive forms, or alternative specialisations. It is commonly assumed that apparently primitive animals represent, at least to some degree, an earlier stage in evolution. But all living species have had equally long to evolve since their common ancestor, and though certain species show greater resemblances than others to early fossil species, it cannot be assumed that features of their biochemistry or physiology also resemble them. Lamprey is such a species, and its haemoglobin shows strikingly different properties from that of mammals. In the oxygenated state it is monomeric, but in the deoxy state it is capable of aggregating to form dimers. This aggregation is the basis for the cooperative properties of lamprey haemoglobin (Briehl, 1963 ; Andersen and Gibson, 1971; Dobi et al., 1973), which seems to be well adapted to the mode of life of the lamprey,

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In the present paper we compare the sequences of lamprey (Li and Riggs, 1970) and Aplysia (Tentori et al., 1973) globins with the postulated vertebrate and molluscvertebrate ancestors to see if differences exist in the rates of evolution of different types of residue positions that could throw light on the relationship of the functions of the molecules with those of their ancestors.

346

B. Tiplady and M. Goodman

Methods and Results The sequence files used were those from the genealogy described previously by Goodman et al. (1975). The nucleotide sequences for Aplysia and lamprey globins together with those of the vertebrate common ancestor and the vertebrate-mollusc common ancestor are set out in Table 1. The nucleotide replacement lengths were calculated for the different types of residue position, using the same functional categories as before. The use of categories derived from mammalian haemoglobin seemed justified in that the tertiary structures of the molecules are sufficiently similar (Hendrickson et al., 1973). These results are presented in Table 2. Between the mollusc-vertebrate ancestor and Aplysia globin, there is no very striking pattern of changes, except that interior positions are relatively conserved. This is not surprising in view of the importance of these positions for maintaining the tertiary structure of the molecule. Between the mollusc-vertebrate ancestor, (i.e. the invertebrate-vertebrate ancestor) and the common vertebrate ancestor the pattern is the same as that described previously (Goodman et al., 1975). In particular, the rate of evolution of the al/32 contact area is high. By contrast, in the evolution of lamprey .haemoglobin from the vertebrate ancestor, there is a high degree of conservation of the al/32 contact site.

Discussion Assuming that high rates of nucleotide substitution in a particular part of the globin molecule represent the evolution of new functions, then it is possible to use functional criteria derived from mamalian haemoglobins for parts of the genealogy which lead to mammalian haemoglobin, or for conservative sites elsewhere. Specific interpretations cannot be made from these categories for rapidly evolving sites in other branches of the genealogy. Thus the most significant result from Table 2 is the conservation of the al/32 contact area in lamprey haemoglobin. This site also shows a high degree of conservation between the vertebrate and/3-a ancestors, after which there is again a rapid rate of evolution in the nascent a and/3 genes. The hypothesis that the homopolymeric association of lamprey haemoglobin is analogous to the al/32 mode of binding was suggested by Li and Riggs (1970). The data of Hendrickson (1970; see also Love et al., 1971) are also in favour of this interpretation. The present data provide additional support for that hypothesis. Moreover the results suggest that an ability to form homodimers by deoxyhaemoglobin had been essentially perfected by the time of the common vertebrate ancestor perhaps 500 Myr ago, and that no further advances in polymer forming ability of haemoglobin occurred in the agnathan lineage descending to present day lampreys. In this respect at least lamprey haemoglobin is a primitive or rather conservative molecule, presumably because stabilising selection in the ecological niche of this agnathan lineage favoured such a molecule. On the other hand, no particularly primitive features are apparent for Aplysia globin, although they may be demonstrated when sufficient functional criteria are available for this molecule.

Primitive Haemoglobin

347

Table 2. Nucleotide replacement lengths for groups of residue positions in the descent of Aplysia and Petromyzon globin Moll-Vert to Aplysia MolI-Vert to Vert Vert to Lamprey Types of Residue Positions Haem contacts in both /3 and czand in myoglobin chains

No. of Replacement Positions Length*

No. of Replacement No of. Replacement Positions Length* Positions Lengths*

17

0.59 (10)

18

O.11 (2)

18

0.39 (7)

Non-haem %/32 contacts in both/3 and ~ chains

9

0.78 (7)

9

0.78 (7)

9

0.11 (1)

Like chain contacts in both/3 and c~chains

2

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3

0

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2

0.50 (1)

%/31 contacts in both/3 and ~ chains

11

0.82 (9)

13

0.23 (3)

9

0.44 (4)

With defined functions in cz but not/3 chains

7

0.43 (3)

7

0.14 (1)

7

0.71 (5)

With defined functions in t3 but not c~chains

12

0.75 (8)

12

0.33 (4)

10

0.30(3)

Remaining interior positions

19

0.32 (6)

19

0.16 (3)

19

0.32 (6)

68

0.62 (42)

67

0.13 (9)

63

0.68 (43)

145

0.59 (85)

148

0.20 (29)

137

0.51 (70)

Remaining exterior positions All positions

* The number of replacements given in the parenthesis divided by the number of residue positions.

References 1. Andersen, M.E., Gibson, Q.H. (1971). J. Biol. Chem. 246, 4790 2. Braunitzer, G., Fujiki, H. (1969). Naturwiss. 56, 322 3. Briehl, R.W. (1963). J. Biol. Chem. 238, 2361 4. Dohi, Y., Sugita, Y., Yoneyama, Y. (1973). J. Biol. Chem. 248, 2354 5. Goodman, M., Moore, G.W., Barnabus, J., Matsuda, G. (1974). J. Mol. Evol. 3, 1 6. Goodman, M., Moore, G.W., Matsuda, G. (1975). Nature 253,603 7. Hendrickson, W.A. (1973). Biochem. Biophys. Acta 310, 32 8. Hendrickson, W.A., Love, W.E., Karle, J. (1973). J. Mol. Biol. 74, 331 9. Johanssen, K., Lenfant, C., Hanson, D. (1973). Comp. Biochem. Physiol. A 44, 107 10. Li, S.L., Riggs, A. (1970). J. Biol. Chem. 245, 6149 11. Love, W.E., Klock, P.A., Lattman, E.E., Padlan, E.A., Ward, J.B. Jr. (1971). Cold. Spr. Harb. Symp. Quant. Biol. 36, 349 12. Tentori, L., Vivaldi, G., Carta, S., Marinucci, M., Mass, A., Antonini, E., Brunori, M. (1973). Int. J. Protein Peptide Res. 5 , 1 8 7 Received April 27, 1976; Revised Version October 22, 1976