Heritage Cases

Being able to access biomolecular information (DNA, proteins) in fossils is a paramount way to accurately determine phylogeny. However, despite the exceptional retrieval of a few Late to Middle Pleistocene (40-700 ky) partial genomes1,2, retrieving ancient DNA (aDNA) is still challenging, notably for the Early Pleistocene. In contrast, proteins are more resistant to post-mortem damage, enabling to consider protein-based molecular investigations even in the deep time (>3 My) and specimens preserved under less favourable conditions3. Introduction Biomolecular information necessary to precise hominin phylogeny at the key period of the genus Homo emergence, between 3 and 2 My, is still sorely lacking. However, in principle proteins resist to post-mortem decay, thus allowing molecular investigation of fossils deep in time, even up to 60 My for some dinosaurs4-6. The recent improvement in sensitivity and resolution of mass spectrometry (MS)-based technologies has facilitated the emergence of paleoproteomics. The in-depth analysis of proteins preserved in ancient biomineralized tissues is now possible even with very limited amount of mineralized fossil material. Ancient proteomes from bone, teeth and dental calculus have revealed high potentials for providing functional information complementary to genetic data7 related to taxonomy8,9, ancient pathogens and (patho)physiology10, diet composition11. Proteins have been sequenced from Pleistocene mammalian and human bone remains12-15. Therefore, as a complement of paleogenomics, paleoproteomics appears as a highly valuable approach for unveiling still contentious aspects of hominin and human evolution7, as protein-based phylogenetic reconstructions is possible even for fossil taxa with no available DNA information9,15,16. Enamel thickness variation stems from an evolutionary interplay between functional/adaptive constraints and strict control mechanisms of the morphogenetic program. This mineralized tissue is routinely considered for assessing dietary adaptions as well as for taxonomic identification17-19. The enamel-dentine junction (EDJ), which originates from the basement membrane, is the primary contributor to the outer enamel surface morphology and represents a reliable taxonomic marker to reconstruct phylogenetic relationships20-22. In addition to morphological information, the hard dental tissues protect the biomolecules from degradation by holding them tightly associated within their mineralized structure. More precisely, the dense hydroxyapatite crystals and tubules forming the enamel and dentine tend to preserve peptides from contamination and degradation12. This is especially true for those tightly associated with the mineralization process and/or tooth structural organization3,6,12. Some tooth proteins (e.g., amelogenin, ameloblastin, enamelin) are very specific to the dental hard tissues, ensuring the reliability of the analyses. Among them, the two forms of amelogenin encoded by sex chromosomes, AMELX and AMELY, offer the opportunity to identify male specimens, a major information in paleoanthropology for assessing the taxon-specific extent of sexual dimorphism23 and sex-related behaviours. Tooth-specific proteins present a number of sequence variations susceptible to discriminate primate taxa, including hominins24. Among them, enamelin is a good candidate to assess enamel thickness variability and dietary adaptive changes among19,25,26. Dental tissues also contain collagens that are considered as pertinent markers for species identification8,15,16, including in hominins27. In addition, polymorphisms in genes or proteins involved in the formation or the constitution of dental mineralized tissue are expected to contribute tooth morphology. For example, non-synonymous polymorphisms in tooth protein enamelin (ENAM), or in the ectodysplasin A receptor (EDAR), have been associated with morphostructural changes such as a thinner vs. thicker enamel26, or with shovel-shaped incisors in Asians28, respectively. This emphasizes the role a single amino acid change in a protein may play in tooth morphostructure. Moreover, we recently provided evidence that some tooth protein variants may be also of major interest for taxonomic discrimination among archaic hominins and modern humans24. Tracking non-synonymous polymorphisms in tooth proteins certainly deserves attention not only for taxonomy, but also to gain insights into the evolutionary relationships between tooth protein polymorphisms and dental morphostructural phenotypes. Understanding molecular evolution in fossil hominins and linking it with the fossil morphological variation is thus a key challenge that awaits to be tackled. For these reasons, dental fossil remains offer serious advantages for the preservation of molecular data for paleoproteomic and paleogenetic studies. While it is not yet evident if aDNA from the Early Pleistocene and earlier periods can be recovered, recent research showed that proteins can survive through time even in hot African environment, as demonstrated by the >3 My peptides entrapped in ostrich egg shell3. The protected and stable temperate environment of some fossil-bearing caves in the "Cradle of Humankind" make them particularly promising sites for the preservation of molecular materials such as proteins, and perhaps even aDNA. Objectives Our objective is to retrieve for the first time biomolecular information from primate tooth fossil remains by applying cutting-edge paleoproteomics technologies. The presence of ancient proteins will be investigated by low-invasive high-performance shotgun mass spectrometry (MS). We expect to provide peptide-based biomarkers for taxonomic determination and sex identification and anticipate that any preserved biomolecular information could significantly help refining the paleobiodiversity and phylogenetic placement of the hominins that lived in South Africa at the period of the emergence of the genus Homo, i.e., Australopithecus and Paranthropus. To the best of our knowledge, the analysis of fossil tooth proteomes is only starting, with most pioneering work focusing on the Eurasian Late Pleistocene and Holocene. By paving the way for a systematic, in-depth analysis of late Pliocene and Early Pleistocene primate remains, this proposal has the potential to represent a major advance in the subtle characterisation of the South African fossil record and to answer still pending fundamental questions on hominin diversity and evolution. Methods The AMIS Laboratory and its close partners at the University of Toulouse (France) are fully equipped and experienced to extract, sequence and analyse fossil biomolecules. Ancient tooth proteins will be extracted at the AMIS laboratory according to state-of-the-art protocols to ensure reliable safety and control procedures enabling to avoid recent contamination of ancient samples by modern and environmental material29,30. Tooth proteomes will be analysed by global shotgun mass spectrometry (MS) within the Proteomics Infrastructure of Toulouse (IPBS, http://proteomique.ipbs.fr/en/front-page/). This approach is able to provide accurate identification and confident validation of in-depth protein content in a single analysis of a tryptic digest of protein extract31-33. Briefly, after washing in SDS 1%, the tooth superficial layer is removed by drilling. Enamel and dentine particles are then harvested above a microtube. This procedure was perfected in our laboratory in order to be minimally invasive (Fig. 1). Fig. 1. Drilling and collecting the dental hard tissue material into a microtube in a completely safe and controlled environment at the AMIS laboratory to avoid contamination. Biochemical procedures based on a filter-assisted sample preparation (FASP) protocol10 are performed to obtain trypsin digested peptides. Tooth peptides mixture is then analysed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) using the latest generation of Orbitrap-based mass spectrometers (Q Exactive Plus and Orbitrap fusion mass spectrometers)31,32,33, followed by bioinformatic analyses using Mascot searches against UniProt and home-made databases in Proteome DiscovererTM. Unlike the largely used ZooMS (zooarcheology by mass spectrometry) approach8 based on collagen peptide fingerprint, the shotgun MS strategy allows in-depth analysis of full proteome with accurate identification and confident validation of hundreds of proteins32,33, even for Pleistocene samples2. Its potential to access ancient physiology and pathophysiology has been already demonstrated7. In order to explore the phenotypic variability of tooth proteins, the protein database (Uniprot) will be hand-upgraded with variants of particular interest for taxonomy, tooth pathologies and dental morphology. The AMIS Laboratory is recognized at the international level for its expertise in the analysis of ancient biomolecules29,30. In our experience, even in ancient samples, a minimum amount of enamel and/or dentine tissue (or the order of a few tens of micrograms), is sufficient to detect specific tooth proteins such as amelogenin, ameloblastin, dentin sialophosphoprotein, enamelinand MMP20, in addition to collagens34,35. Material At a first stage, the material required for the realization of this project is limited to fragmentary fossil cercopithecoid and hominin tooth fragments from the sites of Swartkrans and Sterkfontein. This material is permanently stored at the Ditsong National Museum of Natural History of Pretoria. The selected sample includes five small tooth fragments belonging to cercopithecoid specimens (KA 2351, KA 2352, KA 5334, KA 5243 and KA 5385), as well as hominin dental pieces (SK 835, SK 847 and STS 63) (Fig. 2). In order to be as preservative as possible, we only selected small tooth fragments or enamel and dentine chips to sample here for ancient molecular analyses. In particular, the hominin specimens are already fragmentary or were previously cut for histological analyses (as it is the case for SK 835 for example). This application requests permits to carry out sampling and export the aforementioned samples for protein analyses at the AMIS Laboratory of the University of Toulouse, in France.