garcia olivares & borja 1999 - ancient proteins in fossils from venta micena and cueva victoria

8/6/2019 GARCIA OLIVARES & BORJA 1999 - Ancient Proteins in Fossils From Venta Micena and Cueva Victoria

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Ancient proteins in fossils fromVenta Micena and Cueva Victoria

Enrique GARCA OLIVARES, Concepcin BORJAUnidad de Inmunologa, Facultad de MedicinaUniversidad de Granada

Proteins in fossils from Venta Micena and Cueva Victoria viere analyzed by Lowenstein at the University ofCalifornia, San Francisco with a radioimmunoassay (RIA), and by Borja and Garca-Olivares at the ImmunologyUnit of the University of Granada with an enzyme-linked immunosorbent assav (ELISA). These independentstudies involved the use of polyclonal and monoclonal antibodies against albumin from different species.Theresults from San Francisco (RIA) and from Granada (ELISA) viere compared at the International Conference onHuman Palaeontology, held in Orce in September 1995 (Borja and Garca-Olivares, 1995 and Borja in thisvolume; Lowenstein, 1995). Both sets of data showed that the albumin detected in fossils VM-0 and VM-1960was immunologically more similar to human albumin than to albumin from other species. Lowenstein also foundcollagen and transferrin in VM-0, which were more similar to human proteins, and Borja and Garca-Olivaresdetected IgG in VM-1960 that was more similar to human IgG. Neither group detected human albumin or IgGin fossils CV-1 and CV-2 from Cueva Victoria, assigned to hominids, but both found the reactions for albuminthat would have been predicted from the morphological assignments of the three equid fossils and two bovidfossils, and for IgG in four equid fossils. Not surprisingly, some fossils (two equid fossils and three fossils assignedto hominids) failed to yield proteins. An unexpected result was the finding of human albumin in an equid fossil,probably as a result of external human contamination. This has been used by Palmqvist as the basis for hiscriticism of thc rest of the concordant results from the two groups (Palmqvist, 1997).

PALMQVIST'S CRITICISM OF VENTA MICENA FOSSIL PROTEINSPalmqvist has recently praised the immunological work done by one of us on the proteins in the Venta

Micena fossils (Borja, 1995) in the Bulletin of the Spanish Society of Palaeoanthropology: "...Esta Tesis constituyeun trabajo de investigacin modlico, que marcar un punto obligado de referencia en Paleontologa Molecular..."(This Ph D thesis constitutes a model piece of rescarch work that will be an obligatory reference in MolecularPalaeontology) (Palmqvist, 1996). This contrasts sharply with his current opinion: "...The immunologic analysisof this (VM-0) and other presumably hominid remains froni Venta Micena are far froni being conclusive..."(Palmqvist, 1997) and more sharply with one of lis articles in a local newspaper (Ideal, 15.11.96) "...in viewof his (Garca Olivares) scant, contradictory results and proven lack of scientific method..." ("...a tenor de losescasos y contradictorios resultados obtenidos por el momento en el curso de sus trabajos, salvo contrastar, anms si cabe, su demostrada carencia de mtodo cientfico..."). His criticism can be summarized in the followingpoints:1. The detection of human albumin in a equid fossil from Venta Micena.2. A review paper noting the contradictory results obtained in analyses of blood residues on archaeological

artefacts (Fiedel, 1996).3. The detection of very high amounts of proteins in such very old bones (Venta Micena).4. The absence of IgG in bones buried for 2 months (Cattaneo et al., 1993), which appears incompatiblewith the findings of Borja and Garca-Olivares (1995) of this protein in fossils from Venta Micena.Let us examine these arguments.

AN UNEXPECTED IMMUNOLOGICAL REACTION IN THE FOSSILS FROM VENTA MICENAThe groups in Granada and San Francisco have studied the presence of albumin or IgG in a total of 12morphologically well-matched equine and bovid fossils from Venta Micena (this sample does not include thefossils assigned to hominids), and have detected only one unexpected result: an equid fossil in which a high

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reaction to human albumin was found.This result has been cited as a possible example of accidental contamination(Lowenstein, 1995), an event which is not unsual when dealing with fossil biomolecules, particularly ancientDNA rather than with proteins. Our conclusion is that great care must be taken not to contaminate thespecimens by human contact. Samples, especially the controversial ones, should be tested and retested in differentcenters if possible, and very strict controls must be used. Palmqvist's conclusion is categorical: this singlediscrepant finding invalidates the rest of concordant results.Contamination is very unlikely to have occurred in the equid and bovid fossils in which a horse and bovidprotein respectively were detected, and in which unexpected reactivity for a human protein might be considereda sign of contamination. But, how can we know whether the reactivity for human albumin or IgG detected ina fossil assigned to a hominid was due to external contamination or tu an actual fossil protein still able to providegenetic information? To detect modern contamination in a possibly human bone we have developed the followingprocedure (Borja et al., 1997):The specimen is washed with phosphate buffered salive (PBS) and then treated with a decalcifying solutionof EDTA. Any contaminant protein "attached" to the bone should appear in the PBS extracts. In fossils VM-O,VM-1960 and VM-3691, assigned to hominids, PBS extracts were, however, negative, whereas their correspondingEDTA extracts gave positive reactions for human protein (Borja and Garca-Olivares, 1995 and Borja in thisvolume; Lowenstein et al., submitted). This demonstrated that the protein detected was indeed integrated in themineral structure of the bone, and was not superficial, as would be expected if this protein had originated fromexogenous contamination.

A BONE IS A BONE, NOT A STONEAnother source Palmqvist cites in support of his criticism about our results with fossil bones isa reviewarticle reporting the discrepant results obtained in immunological studies of blood residues of prehistoric stonetools (Fiedel, 1996). Although Palmqvist claims and erroneously reported that Fiedel's paper deals with "...studiesof proteins recovered from fossil bones and blood residues from prehistoric artefacts...", he does not seem to have

read the article carefully, since the paper deals only with prehistoric artefact. We have hardly searched for fossilbonos in Fiedel's article and we have found any.Discrepant results from archaeological stones cannot be extrapolated to fossil bones, in contrast with whatPalmqvist appears to be irnplying. Downs and Lowenstein, 1995 have carried out a series of blind tests of bloodresidues archaeological stones, and in fact, these results are commented in Fiedel's paper: "...Downs' study is, tothe author's knowledge, the only truly objective test that has been conducted to date..." Downs and Lowensteinfound that archaeological blood residues generally could not be identified reliably. Nevertheless, whereas proteinscannot be clearly identified on artefacts only a thousand years old, these biomolecules can survive under the rightconditions and be characterized, in much more ancient fossil bones. Lowenstein detected species-specific collagenand serum factors in fossils as old as a 0.5 Myr-old Horno erectus, a 1.9 Myr-old Australopthecus robustus (Lowenstein,1981) and 8 Myr-old Rarnapthecus and Svapithecus (Lowenstein, 1983). Furthermore, collagen has been foundin a 10-Myr-old unidentified bone (Rowley et al., 1986); osteocalcin has been extracted from 13-Myr-old fossilbovid bones and from 30-Myr-old fossil rodent teeth (Ulrich et al., 1987) and collagen fibrils were observed withelectron microscopy in a 200-Myr-old dinosaur bone (Wykoff, 1972). Moreover, osteocalcin from a bovid fossilretained functionally active gammacarboxyglutamic acid residues after 13 Myrs (Ulrich et al., 1987). These resultsclearly show that proteins from some fossil bones, although fragmentary, can persist and retain enough of theirimmunological properties to provide usable genetic information for periods of milions years.

Preservation seems tobe favored by the fact that proteins are embedded in the mineral phase of the bone,and this provides considerable protection from degradation by environmental conditions. The mineral phase alsoappears to preserve, by diffusion, even the cartilage of some fossil bones (Franc et al., 1995). Obviously thisprotection does not work for proteins of blood residues on archaeological artefacts, since m this case thebiomolecules are not integrated in the specimen, but simply "stuck" to its surface, where they are probably morevulnerable to the effect of environmental agents. For this reason, despite the number of recent reports dealingwith blood residues on prehistoric artefacts, many of them contain conflicting or non-reproducible results. Byconfusing bones and stones Palmqvist commits one of the most common errors in science: the fallacy ofinappropriate extrapolation (Skrabanek and McCormick, 1989).46


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ANCIENT PROTEINS IN FOSSILS FROM VENTA MICENA AND CUEVA VICTORIA

THE NANOGRAM, RATHER A SMALL UNIT

Palmqvist also bases his criticism on "...the detection of verv high amounts of proteins (unexpected in suchold fossils)..." However, Palmqvist provide any background information that would allow the reader to judgewhether the amounts detected in the fossils from Venta Micena are abnormally high. To prove that somethingis abnormally high or low, the figures need to be compared \vith other established measures.Palmqvist's claim makes reference to two parameters: the amount of proteins and the age of the fossils. Isnanogram range too high for 1.6 Myr fossils? Palmqvist seems surprised that4f human albumin wasdetected with RIA in fossils VM-0 andVM-1960 (Lowenstein. 1995). Much lower amounts of albumin in fossils,in the range that Palmqvist would apparently expect to find, would have been below the limits of detection ofthe methods we used to search for proteins. According to Palmqvist's reasoning, any detectable quantity in fossilsfrom Venta Micena would appear to be suspiciously high.We performed quantitative experiments to compare VM-0 and fresh human bone. We found 595 ng ofalbumin/mg of bone in this later bone and 9.8 ng/mg in the fossil (Lowenstein et al., submitted). Moreover,osteocalcin was detected at 5 ng/mg in a 13 Myr old fossil bone (approximately 5 times less than in a recentbone) (Ulrich et al., 1987).Although immunological reactions with fossils give acceptable information about the genetic similarity withmodern species, the amounts of protein found cannot be taken at a face value, since we are obliged to comparefossil proteins that are denatured and broken up, with intact modern proteins. Thus, we have used the terco "ng-equivalents" to denote that the amounts reported are not absolute values (Borja et al., 1997).Many authors agree that there is not strict relation between the biomolecules content and the age of thefossils or ancient bones. In many instances, older bones contain more biomolecules than younger ones (Ulrichet al., 1987; Hagelber et al., 1991). Fossils therefore cannot be judged solely by their age. Other factors suchas the physicochemical enviroment, that have affected the fossil over the time appear to be more important.Nevertheless, these factors are not well established and no general rule for molecular preservation can be appliedto fossils.

No ALL BONES ARE THE SAMEThe last element of Palmqvist's criticism, and the one that gives rise to "...the most severe reservations onthe published reports about protein survival...", is based on a study by Cattaneo et al., 1993. These authorsreported that IgG did not survive in a bone buried for two months. Does Palmqvist conclude that IgG cannotbe preserved for longer than two months in any buried bone, and therefore that it is impossible to detect thisprotein in any fossil? Tuross and Stathoplos (1993) have clearly shown that IgG from fossil bones can be separatedby gel electrophoresis and identified with Western blotting.Is the ELISA used by Cattaneo et al. sensitive enough to detect proteins at very low concentrations?Cattaneo et al. (1990) reported that they detected "...extremely small amounts (10 ng) of protein..." They neededgrams of bone for protein extraction and reported their results in semiquantitative tercos (-,+,++,+++). In ourhands, RIA, ELISA and dot-blotting were able to detect as little as 1 ng or less of protein; moreover, we neededonly few milligrams of fossil bone, and reported results of a more quantitative nature.More importantly, a fossil bone is a unique specimen that becomes a fossil only under very particularconditions. These conditions are responsible not only for the existente of the fossil, but also, probably, for thepreservation of biomolecules. 1.6 Myr-old fossil bones and recent bones buried under garden conditions for twomonths are by no means comparable. Nevertheless, we have also studied buried bones (collected 10 years afterburial a cemetery) and found amounts of albumin not much higher than those detected in fossils (32.6 ng/mg).This apparent contradiction can be explained if we consider that after death, under normal (garden/cemetery)conditions, proteins tends to disappear. However, under the singular conditions that lead to fossilization, as atVenta Micena, proteins are probably "frozen" into the mineral phase of the bone so that they are preserved formillions of years. Palmqvist's attempt to apply to fossil bones conclusions obtained from recent bones is anotherexample of inappropriate extrapolation (Skrabanek and McCormick, 1989). As Poinar pointed out in Nature:"...There are many types of fossilization processes, and to assume that the breakdown of DNA is similar in allof them, or is equivalent to that in non-fossilized material, is not scientific..." (Poinar, 1993)

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BORJA C. (1995). Deteccin y caracterizacin de protenas fsiles mediante tcnicas inmunes. Ph.D d ssertation. Universityof Granada.BORJA C. and GARCA-OLIVARES E. (1995). Detection and characterization of proteins in fossils from VentaMicena and Cueva Victoria by immunological methods. International Conference on Human Palaeontology(Orce, Granada), p. 26.BORJA C., GARCA-PACHECO JM., GARCA-OLIVARES E., SCHEUENSTUHL G. and LOWENSTEINJM. (1997). Immunospecifity of albumin detected in 1.6 million-year-old fossils from Venta Micena, inOrce, Granada, Spain. American Journal of Physical Anthropology, 103: 433-441.CATTANEO C., GELSTHORPE K., PHILLIPS P., and SOKOL R.J. (1990) Blood in ancient human bone.Nature 347: 339.CATTANEO C., GELSTHORPE K., PHILLIPS P., and SOKOL R.J. (1993). Blood residues on stone tools:indoor and outdoor experiments. World Archaol. 25: 29-43.DOWNS E.F. and LOWENSTEIN J.M. (1995). Identification of archaeological blood proteins: a cautionarynote. J. Archaeol. Sci. 22: 11-16.FIEDEL S.J. (1996). Blood from stones? Some methological and interpretative problems in blood residue analysis.Archaeol. Sci. 23: 139-147.FRANC S., MARZIN E., BOUTILLON M.M., LAFONT R., LECHNE DE LA PORTE E and HERBAGED. (1995). Immunochemical and biochemical analyses of 20000-25000-year old fossil cartilage. Eur. J.Biochem. 234: 125-131.HAGELBERG E., BELL L.S., ALLEN T., BOYDE A., JONES S.J. and CLEGG J.B. (1991). Analysis of ancientbone DNA: techniques and applications. Phi/. Trans. R. Soc. Lond. B. 333: 399- 407.LOWENSTEIN J.M. (1981) Immunological reactions from fossil material. P101. Trans. R. Soc. Lond. B 292: 143-149.LOWENSTEIN J.M. (1983) Fossil proteins and evolutionary time. Pontif Atad. Sci. Scr. Var. 50: 151-162.LOWENSTEIN J.M. (1995) Immunological reactions on fossil bones fromLowenstein J.M., Borja C. and GARCA-OLIVARES E. Species-specific albumin in fossil bones from Orce,Granada, Spain. Submitted.PALMQVIST P. (1996). El anlisis de biomolculas fsiles y sus posibilidades en Paleontologa. Boletn de laSociedad Espaola de Paleontologa. 27: 56-61.PALMQVIST P (1997).A critical re-evaluation of the evidente for the presence of hominids in lower pleistocenetimes at Venta Micena, southern Spain. J Human Evol. 33: 83-89.POINAR GO JR. (1993). Reply to Tomas Lindahl: Recovery of antediluvian DNA. Nature 365: 700.ROWLEY M.J., RICH PV, RICH T.H., and MACKAY I.R. (1986) Immunorreactive collagen in avian andmammalian fossils. Naturwissenschaften 73: 620 -623.SKRABANEK P. and MCCORMICK (1989). Follies and Fallacies in Medicine. The Tarragon Press, Glargow, UK.TUROSS N. and STATHOPLOS L. (1993). Ancient proteins in fossil bones. Meth. Enzymol. 224: 121-129.ULRICH M.M., PERIZONIUS W.R., SPOOR C.E, SANDBERG P., and VERMEER, C. (1987) Extractionof osteocalcin from fossil bones and teech. Biochem. Biophys. Res. Commun. 149: 712-719.WYCKOFF R.W.G. (1972) The Biochemistry of Animal Fossils. Bristol: Scientechnica.

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garcia olivares & borja 1999 - ancient proteins in fossils from venta micena and cueva victoria

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