An unexpected finding in prehistoric bison bones

May 19, 2015 § Leave a comment

Cranial bone sample from Bison latifrons (13.009a) with inside surface shown. Presence of material that is consistent with meninges connective tissue is shown. Structure that may be the site of a former blood vessel is found in the upper right of the bottom image (boxed area).

Cranial bone sample from Bison latifrons.

On October 14, 2010, construction workers excavating a site for a reservoir dam near Snowmass Village in Colorado stumbled across bones. The bones belonged to a woolly mammoth. More careful digging revealed close to 5,000 bones from different Ice Age animals. Camels, mastodons, and bison were among them. In a recent paper from the journal Molecular & Cellular Proteomics, researchers reported the analysis of proteins found in the bones of an extinct species of giant bison from the site. From their analysis, they described an unexpected feature of ancient collagen.

The bones at the Snowmass Village fossil site (which is also known as the Ziegler reservoir site) were remarkably well-preserved. The high altitude of the site, which was a lake in the Ice Age, kept it at relatively cool temperatures over the past 130,000 to 150,000 years. The cooler temperatures probably contributed to the preservation the buried materials; even some of the ancient plant material buried at the site was still green at the time of the discovery.

Kirk Hansen at the University of Colorado, Denver, heard of the Snowmass Village discovery in 2010 “while listening to public radio on my way into work.”  Hansen is a protein biochemist whose expertise is in the extracellular matrix. He called the Denver Museum of Nature & Science, which was directing the excavation of the bones, to see if he could help with analyzing samples.

Hansen’s laboratory carries out mass spectrometry analyses and he was aware of existing mass spectrometry work on fossilized proteins. Some studies have suggested that red blood cells can be preserved in ancient bones, but the validity of these interpretations have been questioned.  Skeptics also have wondered about inadvertent contamination of ancient samples with modern proteins.

But, Hansen says, “I thought that the methods we were developing to improve characterization of proteins from the extracellular matrix could be used on these well-preserved samples.” Hansen knew he would get good quality samples from the Snowmass Village site when, he says, “one of the scientists described the smell of the bone fossils as ‘very organic. ’”

Mindful of the issue of contamination, Hansen and colleagues were careful with the samples given to them by the museum. The samples were skull bones from an extinct species of giant-horned bison from the Pleistocene era called Bison latifrons. “We took extra precautions by using new chromatography columns and ensuring the samples were placed in only new vials,” he says.

The investigators carried out mass spectrometry on the proteins left in the bison bones. The biggest challenge was in the data analysis. Some of the proteins had degraded, as expected of old proteins, giving a “laddering” effect in the peptides, and numerous peptides were changed by post-translational modifications.

But the investigators sorted through the data and identified extracellular matrix proteins and plasma proteins. Thirty-three of the ancient bison proteins mapped over to modern bovine proteins, showing the evolutionary kinship.

In particular, Hansen and colleagues sequenced in detail the collagen from the bison samples. The extracellular matrix protein, which forms a fibrous ropelike structure, bore modifications seen in other studies of ancient collagen, such as proline hydroxylation.

But one modification was new and unexpected—hydroxylysine glucosylgalactosylation. “This was the first discovery of a preserved glycan, to the best of my knowledge,” says Hansen. “Finding it in a sample that is over 100,000 years old was surprising.”

Bioarcheologist Matthew Collins at the University of York in the U.K., who specializes in studying ancient collagen, is most impressed with the finding of the hydroxylysine glucosylgalactosylated residue. Glycosylation is a key structural feature of collagen, crosslinking chains together to stablilize its ropelike structure. But it was assumed the seemingly labile glycosylated residues would not withstand the test of time.

“You’d imagine, over this period of time, you would have lost the sugars. That’s one of the reasons why we never bothered to look for them: We didn’t expect to find them. This work elegantly shows that I was wrong!” says Collins. “We’re now going back and looking at our samples for glycosylated residues.”

As Hansen and colleagues were working on the bison samples, data came from a young Siberian woolly mammoth called Lyuba. Her proteins bore similar modifications to those of the bison proteins.  “Finding these modifications in modern tissue samples usually requires some form of enrichment,” says Hansen. But, with these two fossils, “the modifications were relatively easy to find.” He says the discoveries suggest that collagen with hydroxylysine glucosylgalactosylation might be enriched over time because it creates a stable complex.

Hansen and his team’s next aim is to study the relationships between collagen modifications and collagen fiber architecture. The ramifications of the work will go beyond the study of ancient proteins. As Hansen explains, “Once we make progress in this area, we will have a better understanding of the microenvironment’s role in tumor progression and the ability to rationally design biomaterials for tissue engineering applications.”

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