1. Mol. Biol. (1967) 26, 351-352
to the Evolution of Collagen
Comparison of phylogenetically different collagen proteins on the basis of primary structure determination is thus far not feasible. Amino acid sequence analysis is rendered difficult by certain structural peculiarities, e.g. high imino acid content in the so-called apolar regions, and a molecular weight of approximately 300,000; some sequence rules, however, are known which govern and characterize the structure of the molecule (Hannig & Nordwig, 1967). One of these properties, a regular alternation of concentrations of polar and apolar amino acids, respectively, documents itselfin the cross-striation pattern of the electronmicroscopic picture. Thus, electron micrographs of the so-called long spacing segments, an artificial quaternary structure of collagen, provide a kind of blueprint for the array of t’he amino acids in the molecule---a case unique in protein chemistry. We have been using this technique for comparing the gross structure of the collagens of highly developed mammals and of lower invertebrates. We isolated native soluble collagen (actinocol) from the coelenterate Actinia eqzcina L., a kind of sea anemone, by acidic extraction at low temperatures. Long spacing segments were precipitated by dialysis against ATP solutions. An electron micrograph of such a preparation of actinocol is shown in Plate I (upper part) and compared with a segment from calf skin collagen (Plate I, lower part) obtained by an analogous procedure. There is no difference in the cross-striation pattern of the segments of both collagena, as concluded from the number of the striations and their relative positions (and apparently also from their relative intensities). This certainly does not mean that the amino acid sequence is actually identical in both cases; there is sufficient evidence (Maser & Rice, 1962; Josse & Harrington, 1964; Nordwig & Hayduk, manuscript to be published) that even highly purified preparations of invertebrate collagen differ in amino acid composition from calf skin collagen. Also, vertebrate collagens may differ from one another in this respect, especially due to differences in the environment of the original animal (see Josse & Harrington, 1964, for further references). In any case, our finding apparently means that the individual polypeptide chains forming a collagen molecule are homologous, as can already be deduced from the results of Kuhn et aE.(Kuhn, Tkocz, Zimmermann & Beier, 1965; Kuhn, Zimmermann $ Tkocz, 1967). In other words, all the features of the primary structure responsible for the helical and quaternary array of the molecule must be the same for both calf skin collagen and actinocol. This is surprising considering the pronounced separation in phylogenetic development of the two animals compared. From this finding we conclude that only the rigid rod molecule of collagen made up from three strands of poly-n-proline II structured cc-chains (Rich & Crick, 1961; Ramachandran & Sasisekharan, 1961; Ramachandran, 1963) along with its characteristic charge pattern is capable of fulfilling its task as the main protein of the connective and skeletal tissue in the animal kingdom. Variations of its amino acid composition by mutation, deletion or insertion during evolution have been limited by these fundamental structural requirements of the tertiary and quaternary structures, i.e. by 361
structural proteins the biological function of the molecule. In this regard, “inert” correspond to the biologically more active globins, e.g. enzymes (Acher, 1966). It should be stressed that the length of the molecule seems to be constant in both collagens investigated (2800 a), This fact again suggests a rather low degree of mutability of the collagen (or collagen a-chain) cistron during evolution. Details on our work regarding invertebrate collagen are to be published. ARNOLD NORDWIG URSULAHAYDW
Max-Planck-Institut ftir Eiweti- und Lederforschung Miinchen 15, Germany SchillerstraBe 46 Received
Acher, R. (1966). Angew. Chemie, 78, 856. Angew. Chemie, Internat. Ed. 5, 798. Hannig, K. & Nordwig, A. (1967). In Treatise on, Collagen, vol. 1: Chemistry of Collagen, ed. by G. N. Ramachandran. New York: Academic Press, in the press. Jesse, J. & Harrington, W. F. (1964). J. Mol. Biol. 9, 269. K&n, K., Tkocz, Ch., Zimmermann, B. & Beier, G. (1965). Nature, 208, 685. Kiihn, K., Zimmermann, B. & Tkocz, Ch. (1967). Int. Symp. Conformation of BiopoEymers, Madras/India. New York: Academic Press, in the press. Maser, M. D. & Rice, R. V. (1962). Biochim. biophys. Acta, 63, 255. Ramachandran, oi. N. (1963). In Aspects of Protein Structzcre, ed. by G. N. Ramachandran, p. 39. New York: Academic Press. Ramachandran, G. N. & Sasisekharan, V. (1961). Nature, 190, 1004. Rich, A. & Crick, F. H. C. (1961). J. Mol. Biol. 3, 483.