We have previously shown that the thermal stability of animal collagens correlates with the number of hydrophobic amino acid residues in their composition: the more hydrophobic residues in a molecule, the higher the denaturation (melting) temperature of collagen (Efimov, 1987, Meshcheryakova et al., 2024). Analyzing this dependence, we found that there are differences between warm-blooded and cold-blooded organisms. In particular, with the same proportion of hydrophobic amino acid residues, the thermal stability of collagen in warm-blooded organisms is several degrees higher than in cold-blooded organisms (Fig. 1). One of the main objectives of this study is to establish what are the structural differences between collagen molecules from warm-blooded and cold-blooded organisms.

As we discussed in our previous study, collagen denaturation temperatures range widely from 5 to 6 °C in Antarctic fish to 45-46 °C in hydrothermal vent polychaetas (Rigby, 1968, Gaill et al., 1995). The molecular mechanisms that determine such wide limits of collagen thermal stability are not fully understood.

The triple helix of type I collagen contains three types of chains (subunits) α-1, α-2 and α-3. Subunits α-1 and α-2 are found in animals of various phylogenetic and ecological groups. Subunit α-3 is found only in cold-blooded organisms − some fish species (tilapia, pollock, rainbow trout, hake, eel, carp, grass carp, etc.) and some invertebrates (octopus, squid, paper nautilus and jellyfish) (Kimura and Ohno, 1987, Saito et al., 2001, Nagai, 2004, Hofman and Newberry, 2011). Collagen type I of most warm-blooded and cold-blooded animals is represented by the heterotrimer 2α1(I):α2(I), in some cases by the homotrimer 3α1(I). Among fish and invertebrates, in addition to the above trimers, the heterotrimer α1(I):α2(I):α3(I) may be found. In contrast to the amino acid composition of collagens, the amino acid composition of subunits has been studied very limitedly (Kimura and Ohno, 1987, Ramshaw et al., 1988, Saito et al., 2001, Zeng et al., 2012, Weng and Wang, 2018). The few studies that do show that subunits of different types have different amino acid compositions, which presumably affects the thermal stability of collagen.

Data on comparative studies of the temperature stability of various subunit compositions of collagen are very limited due to the existing species and tissue differences in the thermal stability of collagens. A number of data have been obtained for different organs, for example: the collagen of the caudal fin of the Japanese sea bass with the subunit composition 3α1(I) has a denaturation temperature 1.5 °C higher than the collagen from the skin of this fish with the composition 2α1(I):α2(I) (Nagai and Suzuki, 2000, Nagai, 2004). Certain results have been obtained in the study of artificially bred homozygous and wild heterozygous mice, which have homotrimers of 3α1(I) and heterotrimers of 2α1(I):α2(I) collagens, respectively (Kuznetsova et al., 2003). The authors have shown that the denaturation temperature of collagen 3α1(I) of homozygous mice is 2.5 °C higher than that of collagen 2α1(I):α2(I) of heterozygous mice. The authors concluded that the presence of the α2 chain in collagen makes a certain contribution to the decrease in its thermal stability and that the differences in thermal stability are due primarily to differences in the amino acid composition of the α1 and α2 chains. As for the thermal stability of the α3 subunit, it is assumed that it has lower thermal stability than the α1 subunit. For example, collagen from the swim bladder of pollock 2α1(I):α2(I) has a denaturation temperature two degrees higher than pollock skin collagen with the composition α1(I):α2(I):α3(I) (Kimura, Ohno, 1987). Similar results were obtained for the collagen of the skin and muscles of skipjack tuna and rainbow trout (Zhu and Kimura, 1991, Saito et al., 2001). Experimental data on the amino acid composition of the three subunits show a lower content of hydrophobic amino acid residues in the α2 and α3 subunits compared to the α1 chain (Kimura and Ohno, 1987, Ramshaw et al., 1988, Kimura and Matsui, 1990). Thus, different amino acid compositions of the subunits and the molecular composition of the triple helix will determine the thermal stability of collagen.

A comparative study of the amino acid composition of collagen sequences in various animals will help to understand the general and specific mechanisms underlying the stability of collagens in warm-blooded and cold-blooded organisms, how they are realized under different temperature conditions, and how the subunit composition of collagens can affect the thermal stability of triple helices.

In this work, we analyzed the amino acid compositions of the α1, α2, and α3 chains of type I collagen from mammals, birds, reptiles, amphibians, fish, and invertebrates collected in the UniProt database. In addition to traditional warm-blooded and cold-blooded organisms, it was interesting to study the sequences of organisms with a non-standard type of thermoregulation. Among the sequences found, there were only the naked mole rat, a mammal leading an underground lifestyle and belonging to ectotherms, and bluefin tuna, whose thermoregulatory muscle activity ensures a constant body temperature and belonging to the group of homeotherms. The aim of the work was to compare the amino acid composition of subunits belonging to type I between warm-blooded and cold-blooded organisms and to compare the amino acid composition of different types of subunits with each other.