Identification of the earliest collagen- and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)

Mortuary practices in human evolution record cognitive, social changes and technological innovations. The Neolithic Revolution in the Levant was a watershed in this domain that has long fascinated the archaeological community. Plaster modelled skulls are well known at Jericho and several other Neolithic sites, and in Nahal Hemar cave (Israel, ca. 8200 −7300 cal. BC) excavations yielded six unique human skulls covered with a black organic coating applied in a net pattern evoking a headdress. This small cave was used as storage for paraphernalia in the semi-arid area of the Judean desert and the dry conditions preserved other artefacts such as baskets coated with a similar dark substance. While previous analysis had revealed the presence of amino acids consistent with a collagen signature, in the present report, specific biomarkers were characterised using combined proteomic and lipid approaches. Basket samples yielded collagen and blood proteins of bovine origin (Bos genus) and a large sequence coverage of a plant protein charybdin (Charybdis genus). The skull residue samples were dominated by benzoate and cinnamate derivatives and triterpenes consistent with a styrax-type resin (Styrax officinalis), thus providing the earliest known evidence of an odoriferous plant resin used in combination with an animal product.

Outline of the cave, showing the approximate positions of finds and the skulls near the Western wall (one square represents 1 m 2 ).

Amino acid distribution obtained by RP-HPLC
Collagen is the major protein identified in archaeological bones and skin. It is a fibrous protein found in skin, sinews, and bone and consists of a coil of three chains (two identical chains α1 and one α2), forming a triple helix where each chain is made of repetitive glycine-Xaa-Yaa motifs where Xaa and Yaa can be almost any other amino acid but is mostly proline or hydroxyproline and Yaa respectively. As such, glycine accounts for approximately one third of the amino acid content, giving it a distinctive amino acid composition signature.     Profiles obtained by pyrolysis gas chromatography Figure S4: Py-GC/MS total ion current chromatograms of A) NH2824, B) NH2825, C) NH2826, D) NH2868 and E) NH2869. Compound numbers refer to those reported in Table  S2.     Most proteins identified are from Human and most likely contamination. The cytoskeletal keratins identified are common laboratory contamination from dead skin resulting from handling. An indication that the proteins are likely false positives is the complete absence of Asn and Gln deamidation (a modification commonly identified in ancient proteins) in the proteins identified in the skull while deamidation is consistently found in the proteins identified in the basket.

Identification of the triterpenoids occurring in samples NH2968, NH2969 and in the resin of S. officinalis
The structural identification of the triterpenoids as C-6 oxygenated derivatives of oleanolic acid in samples NH2968 and NH2969 and in the fresh and oxidized resin of S. officinalis is mainly based on mass spectral investigations using GC-MS (electron impact (EI), chemical ionization (CI) and field ionization (FI) modes) as well as on comparison with published mass spectra of triterpenoids from Styrax sp 10 or other plant species 11 and of amyrin-related epoxylactones 12 .

Taxonomy of Charybdis maritima (Drimia maritima)
The Drimia belongs to the Urgineoideae subfamily of the Hyacinthaceae family (hyacinth family) which contains three other subfamilies (Hyacinthoideae, Ornithogaloideae and Oziroeoideae). According to Pfosser and Speta (2004) 15 , the sea onion, well known since Antiquity for its medicinal properties and by the Romans as Scilla, was first wrongly attributed to the Scilla genus in the Hyacinthoideae subfamily. The genus Urginea (Urgineoideae subfamily) was created in the 1830s and was followed by the inclusion by Baker of the sea onion in 1873.
The genera Bowiea and Drimia were then added and Charybdis created later, in which these authors (Pfosser and Speta (2004) 15 ) have classified the Mediterranean squills.

Charybdin
Charybdin was characterised and named by Touloupakis et al. (2006) 16 as a novel 29 kDa type I ribosome-inactivating protein (RIP) found in bulbs of C. maritima. There are seven other ribosome-inactivating proteins characterized in the Hyacinthaceae family; of them the closest species has less than 50% percentage coverage in common with charybdin.
RIPs inhibit protein synthesis by enzymatically damaging ribosomes; there are of two types, type I has a single peptide chain, type II contains two. By cleaving a certain site in rRNA, they stop protein synthesis, thus inhibiting ribosome activity 17 . Some RIPs such as ricin are potent toxins. In C. maritima, Touloupakis et al. 16 mention that with 150-200 mg per 100 g, the "bulbs contain extremely high quantities of the charybdin protein. The initial extract contained mainly charybdin and very small amounts of other proteins, which were only observed when the gel was overloaded." Charybdin was the only protein characterised in this study. At the time of writing, there are 265 entries available in NCBI (http://www.ncbi.nlm.nih.gov/) for the Urgineoideae sub-family, 174 entries for the genus Drimia/Charybdis and 11 for D. maritima (mostly chloroplast proteins).
In addition to characterising charybdin, the study highlights a substitution at position 79 in the active site of RIPs where tyrosine is replaced in charybdin by valine. The authors argue that this substitution might be responsible for its low inhibitory activity compared with other RIPs, and that the protein (as the main protein constituent in the bulb) might have a different function such as storage. The peptide which bears Val79, DDLVLR, was successfully identified with a score of 49, as well as IHRDDLVLR with a score of 42. While the first peptide is highly conserved in plants and bacteria (but not identified in RIPs), the second one is found only in a handful of bacterial species. Figure S12: The theoretical distribution in amino acids (relative distribution of the amino acids identified by RP-HPLC) in the charybdin protein from Charybdys maritima (RIP_DRIMA) is plotted against the amino acid analysis distribution obtained in the bone reference and sample NH2825. In the hypothesis that charybdin is the sole plant protein constituent in the organic residue of NH2825, it would require twice as much charybdin as collagen to bring the concentration in glycine from its level in bone collagen to the one detected in NH2825.