By Vivienne Baillie Gerritsen
‘What’s a fossil Mum?’ A question to which most Mums would answer, ‘A fossil, sweetie, is bone which has become stone because it’s been lying somewhere for a very very long time.’ The process of bone diagenesis is just that little more complicated but on the whole Mum’s answer is not incorrect. At least…this is what we have always been taught. However, before the bone becomes absolute stone, some organic parts - depending on the environment - can actually survive quite a long time. Millions of years actually. Scientists have already managed to extract DNA from fossil bone - though in terrible shape. A pity, because DNA, though minute, can stash huge amounts of information. Something not quite so delicate had to be found; something which could also offer biological information from the past. A protein perhaps? Yes. In the 1980s, the existence of a bone protein, osteocalcin, was detected in bovid bones dating back 13 million years and rodent teeth dating back 30 million years! In the 1990s, osteocalcin was detected in 75 million-year-old fossils such as the duck-billed dinosaur. The tricky part was to extract the protein from the bone intact and in sufficient quantities to be able to sequence it. This was only achieved a decade later in fossil bison bone. But what an achievement. And little did osteocalcin know that it would become such a famous molecule.
Bone is an exceptional organic tissue in that it is largely mineral (70-90%). The organic part is mainly collagen. Noncollagenous matrix proteins make up for the rest, of which osteocalcin represents just a tiny fraction. What an interesting fate for such a humble protein! Osteocalcin is a small protein, barely 50 amino acids long. Also known as the bone Gla-protein, because it has a number of g-carboxyglutamic acid (Gla) residues in its sequence. It is found in bone only, secreted there by osteoblasts, where it binds to the bone minerals. And it is the tight bonds it makes with the bone minerals, which most probably protect it from vanishing in the process of fossilization. It is directly associated with bone formation and mineralization - though in what way exactly still remains unclear. What has been discovered is that impaired osteocalcin expression causes bone calcification to spread into nearby cartilaginous structures, so it must somehow have a role in directing and controlling bone formation.
The osteocalcin that became a star was extracted from fossilized bison bone, Bison priscus, which was radiocarbon-dated back almost 60'000 years. Bison priscus, more commonly known as the steppe bison, though now extinct is quite well known thanks to prehistoric paintings in Paleolithic caves and fossils found in permafrost - in particular, a near intact carcass of an 8 to 9 year-old male carcass found in 1979 in Alaska and known as ‘Blue Babe’. ‘Blue’ because the specimen was almost entirely coated with vivianite, a blue iron-phosphate, and ‘Babe’ from the North American tales of Paul Bunyan, a lumberjack who once brought up a blue ox… Steppe bison stampeded the steppe-like grasslands of northern Eurasia and North America during the Pleistocene (2 million to 10'000 years ago); the bone fossils from which were extracted the to-be-sequenced osteocalcin were found in Alaskan and Siberian permafrost.
Fig. 1 One of the bison drawn in the Altamira Cave in Spain*
The great breakthrough is that osteocalcin was extracted from crushed bone fossil and ultimately fully sequenced, which is something that had met with hopeless failure until the year 2002. How was it done? Small amounts (20 mg) of osteocalcin were extracted thanks to techniques to which molecular geneticists are largely accustomed and are used for DNA purification, not protein purification. Until then, large amounts of protein were needed but were very difficult to come by. This solved the ‘quantity’ problem. The sequencing was performed thanks to a technique (matrix-assisted laser desorption ionization mass spectrometry) which, unlike sequencing by Edman degradation, is not hindered by amino-terminal blockage. Thus solving the ‘sequencing’ problem. As a result, the protein was fully sequenced and the ancient bison protein was found to match the modern osteocalcin bison protein. It even predicted the single amino-acid substitution which makes the difference between cow and bison osteocalcin sequences.
Fig.2 The fossil B.priscus osteocalcin sequence appears above mass spectra.
What’s the point of it all? Well…though DNA is far more informative from an evolutionary point of view than protein, and it is also far easier to sequence, it is also far more subject to lab contamination. What’s more, DNA survives less well than protein in the process of fossilization. It has been estimated that protein could survive millions of years (whilst DNA being a more fragile molecule seems to survive only thousands) - an exciting prospect since this could take us right back to the beginnings of human evolution. The key now would be to seek for proteins which are even more informative from an evolutionary and perhaps even animal behavior point of view. Besides paleontology, studies on the survivability of proteins - i.e. protein degradation - are obviously of great interest to those who work in the field of forensics. What is more, further research into osteocalcin itself could ultimately inspire the design of drugs for the treatment of bone diseases such as osteoporosis, in which osteocalcin may well have a role.
Cross-references to Swiss-Prot
P83489: Bison priscus (steppe bison) osteocalcin
References
1. Nielsen-Marsh C., Ostrom P.H., Gandhi H., Shapiro B., Cooper A., Hauschka P.V., Collins M.J.
Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older than 55ka
Geology 30:1099-1102(2002)
2. Nielsen-Marsh C.
Biomolecules in fossil remains
The Biochemist: 12-14(2002)
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