Bone protects proteins over thousands of years Extraction analysis and interpretation of extracellular matrix proteins in archeological skeletal remains.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 123:30 –39 (2004) Bone Protects Proteins Over Thousands of Years: Extraction, Analysis, and Interpretation of Extracellular Matrix Proteins in Archeological Skeletal Remains Tyede H. Schmidt-Schultz1 and Michael Schultz2* 1 2 Department of Biochemistry, University of Göttingen, Göttingen D-37075, Germany Department of Anatomy, University of Göttingen, Göttingen D-37075, Germany KEY WORDS intact ancient bone-matrix proteins; Western blot; 2-D electrophoresis; proteome analysis ABSTRACT In a good state of preservation, bone conserves the entire protein pattern of extracellular bone matrix proteins over thousands of years. The quality of the proﬁles of matrix proteins isolated from ancient bones (ranging from the pre-Pottery Neolithic Phase to Early Modern Times from different archaeological sites in different geographical areas), separated by electrophoresis, is as good as those from recent bones. Molecules arising from collagenous proteins (e.g., collagen type I), from the noncollagenous group (e.g., osteonectin), and from the immune system (e.g., immunoglobulin G) were identiﬁed in Western blots by speciﬁc antibodies. A comparative study of the immunoglobulin G content of the bones of ﬁve pre- historic children showed the lowest immunoglobulin G content in a child who suffered from chronic scurvy. Ancient bone proteins were also separated by two-dimensional electrophoresis. This technique makes fractionation of the complex protein mixtures of extracellular bone matrix more reproducible. Bone retains a chemical memory of earlier metabolic stimuli in its conﬁguration of collagenous and noncollagenous proteins. In combination with the results of the microscopic examination of ancient bone, it should be possible to obtain more reliable information on the history and the evolution of diseases, based on analysis of intact proteins. Am J Phys Anthropol 123: 30 –39, 2004. © 2004 Wiley-Liss, Inc. Among the organs of most vertebrates, bone is unique in both structure and function. Bone is a biphasic system consisting of an organic matrix in intimate contact with a microcrystalline mineral phase. It is from this combination that bone derives the strength necessary to function both as a supportive framework for the body and as mechanical points for muscle attachment. The well-organized network of bone extracellular matrix (ECM) consists of about 90% collagens and about 10% noncollagenous proteins (NCPs). There is tremendous diversity among the NCPs. A variety of NCPs has been identiﬁed, such as ﬁbronectin, osteocalcin, osteopontin, osteonectin, bone sialoprotein II, decorin, and biglycan (Fisher et al., 1987; Hauschka et al., 1987). Of this group, only osteocalcin and bone sialoprotein II are speciﬁc for bone, whereas the other proteins are also present in other noncalcifying tissues. At present, our knowledge of their function and, particularly, of their effect on bone metabolism is limited. In general, it is thought that these proteins play a role in the regulation of the mineralization process (Boskey, 1996; Roach, 1994). A second group of NCPs consists of growth factors stored in the bone matrix (Centrella et al., 1994). For these proteins, distinct cellular effects have been demonstrated, such as regulation of growth and differentiation of cells. Examples include trans- forming growth factor-␤ (TGF-␤), insulin-like growth factors, and bone morphogenic proteins. Interactions are present between the two groups of NCPs. Bone sialoprotein II and osteopontin increase intracellular calcium in osteoclasts (Pannicia et al., 1992). Decorin was shown to bind TGF-␤ and enhances its activity (Takeuchi et al., 1994). TGF-␤ modulates osteopontin and osteocalcin production (Centrella et al., 1994; Staal et al., 1996). At present, it appears that both groups of NCPs are involved in bone remodeling and fracture healing. It is likely that for optimal regulation of bone metabolism, they act in concert (Derkx et al., 1998). Knowledge of all bone matrix components is a prerequisite for the understanding of the biochemistry and physiology of bone. This is also the basis for discussion on diseased states of bone. Bone, particularly its compact component, protects the mole- © 2004 WILEY-LISS, INC. *Correspondence to: Michael Schultz, Zentrum Anatomie der Georg-August-Universität, Kreuzbergring 36, D-37075 Göttingen, Germany. E-mail: firstname.lastname@example.org Received 10 October 2002; accepted 28 February 2003. DOI 10.1002/ajpa.10308 31 INTACT BONE PROTEINS IN SKELETAL REMAINS TABLE 1. Samples taken from recent bones No. Bone Sex Age (years) 1 Right femur Male 76 2 Occipital bone Male 86 3 Right femur Female 93 Preservation Source Frozen directly after dissection Formalin-ﬁxed over 1 month Formalin-ﬁxed over 1 year Pathology, Department of University of Göttingen, Germany Pathology, Department of University of Bochum, Germany Anatomy, Department of University of Göttingen, Germany TABLE 2. Samples taken from ancient bones No. 1 2 3 4 5 6 7 8 Bone Sex Age (years) Archaeological site Chronology Left femur Right femur Right femur Left humerus Right humerus Left parietal bone Left parietal bone Right humerus Male Female Child Child Child Infant Infant Child 35–40 21–25 12–13 12–14 14–16 6–8 months 2–3 8–12 Gottorf, Germany Barbing, Germany Bajkara, Kazakhstan Gemeinlebarn, Austria Franzhausen, Austria İkiztepe, Turkey İkiztepe, Turkey Basta, Jordan AD 1600 EMT AD 450–700 EMA 800–500 BC IA (Scythian) 2200–1800 BC EBA 2200–1800 BC EBA 2400–2200 BC EBA 2400–2200 BC EBA 7500–6000 BC Late PPNB cules inside the ECM after the death of an individual much better than in other organs. Therefore, it should be possible to extract all these above-mentioned proteins from dry, macerated specimens. In a good preservation state, bone conserves intact collagen molecules and many, if not all, NCPs from recent and ancient times. NCPs in living tissues are bound very tightly to apatite. The greatest problems to be overcome are how to detach proteins from the hydroxyapatite, and how to avoid decomposition of the proteins during the extraction process. The detection of enzymes in ancient Egyptian mummies presents very similar problems in the procedure of protein extraction and detection. In the case of the mummy of Idu II, who lived in the Old Kingdom of Egypt (ca. 2200 BC), morphological and microscopic results (Schultz, 1996) demonstrated that this mummy was partly deﬂeshed, especially in the region of the trunk (e.g., the pectoralis muscle). These results were conﬁrmed by Weser et al. (1998). The GC/MS spectrogram of the clavicle of Idu II showed a high concentration of embalming components; this bone could only have been invaded if there had been direct contact with the bone surfaces. Thus, skin and superﬁcial muscles were apparently partly cut away during the mummiﬁcation process. Others also demonstrated the potential of ancient human bone matrix (Etspüler et al., 1995, 1996; Weser et al., 1996). They isolated, immunologically and biochemically, active alkaline phosphatase from well-preserved mummiﬁed bones (Etspüler et al., 1995, 1996) and from nonmummiﬁed bones (Weser et al., 1996). Alkaline phosphatase is an enzyme with a molecular weight of about 200 kD. The presence of alkaline phosphatase activity suggests the preservation of some tertiary structures, resulting in the preservation of active sites and/or immunoreactive epitopes. The purpose of this paper was to demonstrate the signiﬁcance of collagenous and noncollagenous bone matrix proteins extracted from archaeological skel- etal remains for paleopathology and bioarchaeology. Using the technique presented in this paper, the matrix proteins are better solubilized than by other techniques. Therefore, the separation of proteins is improved, and there is a better guarantee of identiﬁcation by speciﬁc antibodies. The combination of protein chemistry with histomorphological techniques (Schultz, 1986, 2001b) will make diagnoses of diseases in ancient skeletal remains more reliable. MATERIALS AND METHODS Materials The specimens used for this study were carefully selected; samples affected by decomposition and diagenesis were strictly ruled out (Tables 1 and 2). Recent samples taken from long bones and the skull (Table 1) which had been frozen directly after sectioning (Pathology Department) or ﬁxed in formalin (Anatomy Department) were investigated. For comparison purposes, individuals were chosen who were older than 70 years and who had died without any speciﬁc disease. The ancient bone samples were taken from adult and subadult individuals; only very well-preserved specimens were chosen for study. Preservation was checked by microscopic techniques (thin ground sections viewed in plane and polarized light; scanningelectron microscopy). All burials were inhumations which had not been disturbed in ancient or modern times. Different time periods were chosen to see whether very old specimens were also suitable for such an investigation. For comparison purposes (with the long bone samples of two recent individuals; see Table 1, nos. 1 and 3), long bone samples of two adult individuals were selected. One of these individuals dated from the Early Middle Ages (EMA), and the other from Early Modern Times (EMT) (Table 2, nos. 1 and 2). Additionally, a small series of infants and children was chosen dating from the Iron Age (IA) (Table 2, no. 3) through the 32 T.H. SCHMIDT-SCHULTZ AND M. SCHULTZ Early Bronze Age (EAB) (Table 2, nos. 4 –7) to the earliest, i.e., pre-Pottery Neolithic B (PPNB) (Table 2, no. 8). Methods Preparation of thin ground sections for microscopic research. After documentation (photos, drawings, and measurements), thin ground sections of bone samples were prepared by suitable techniques (Schultz, 1988a; Schultz and Drommer, 1983). Cleaning and degreasing of bones. The external surfaces of samples taken from human long and skull bones were cleaned mechanically and treated in a buffer (50 mM Tris, 50 mM E-aminocaproic acid, pH 9.1) in an ultrasonic bath. About 1 mm of the periosteal and the endosteal surfaces was scraped off with a scalpel blade. Degreasing is employed mainly for recent bone samples. Cleaned bone samples were washed for 1 day in CHCl3 with permanent stirring; remains of CHCl3 were removed by washing in an ultrasonic bath ﬁlled with autoclaved bidistilled water. After complete drying, the bone was stored at ⫺20°C. Bone powdering. Only the compact substance (compacta: external and internal lamina of skull vault) was reduced to powder to be used in the analysis of samples. Bone samples, cooled in liquid nitrogen, were broken into small pieces with a cooled electric saw. Alternating sawing (30 sec) and cooling pulses (2 min) of the bone were performed in liquid nitrogen. Powdering of small bone pieces was achieved under permanent cooling in liquid nitrogen in the Mikro-Dismembrator U (B. Braun Melsungen AG) at a shaking frequency of 1,600 rpm for 2 min. Extractions of bone matrix proteins (modified according to Termine et al., 1980). About 1 g of mineralized bone powder was extracted with 5 ml buffer A (4 M guanidine-HCl, 20 mM NaH2PO4, 30 mM Na2HPO4, pH 7.4) under permanent stirring for about 1 day (4°C). After centrifugation (10,000g, 30 min, 4°C), the supernatant was removed. The pellet was extracted with 5 ml buffer B (buffer A and 300 mM EDTA) under constant stirring for 1 day (4°C). From the resulting extract, chelated calcium ions were removed by washing (35,000g, 20 min) three times with autoclaved bidistilled water. The pellet, containing the bone matrix extract (BME) was lyophilized. The BME can be stored for several months at ⫺20°C. Each extraction step was carried out with a combination of protease inhibitors, 5 mM benzamidine, 1 mM PMSF, 2 mM aprotenin, 50 M leupeptin, and 10 mM EDTA. All procedures were performed with gloves, autoclaved instruments, and autoclaved or sterile ﬁltered solutions. Solubilization, precipitation, and separation of proteins. About 20 mg of BME were sonicated twice in ice for 7 sec in a neutral 50 mM phosphate buffer with the protease inhibitors 2 mM aprotenin, 1 mM benzamidine, 10 mM E-aminocaproic acid, and 10 mM EDTA. After solubilization, proteins were precipitated with TCA (8% end concentration) and separated by electrophoresis using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), according to Laemmlie (1970). Protein determination (Bensadoun and Weinstein, 1975). This is a modiﬁed “Lowry procedure” (Lowry et al., 1951), with which proteins can also be assayed in the presence of interfering chemicals. The protein levels can then be estimated quantitatively for the precipitated protein by the standard “Lowry procedure” (Lowry et al., 1951). Western blot analysis. Nondenatured protein samples (collagen type I) or denatured protein samples (IgG and osteonectin) were separated by SDSPAGE (6% T and 2.5% C for collagen type I; 10% T and 2.5% C for IgG and osteonectin) and transferred to polyvenylidene ﬂuoride (PVDF) membranes. The ﬁrst antibody, mAB against human collagen type I from mouse, dilution 1:1,000, AB against the heavy chain of human IgG or against human osteonectin from rabbit at a dilution of 1:1,000, was applied. As secondary antibodies, horseradish peroxidaselinked anti-mouse or anti-rabbit was used at a dilution of 1:40,000; bands were visualized by the enhanced chemiluminescence (ECL)-Plus detection system. Silver staining. All silver staining methods depend on the reduction of ionic to metallic silver to provide metallic silver images. The silver stain protocol according to Swain and Ross (1995) was employed. Two-dimensional electrophoresis. Solubilized proteins were loaded onto a dehydrated immobilized pH gradient (IPG) strip (7 cm, pH 4 –7). Focusing was performed at maximally 20°C (Protean IEF-cell, BioRad) (4,000 volts, 10,000 Vh). After focusing, strips were incubated with 2% DTT in equilibration buffer (6 M urea, 0.375 M Tris, 2% SDS, 20% glycerol) for 10 min to reduce the proteins. These were then carbamidomethylated for a further 10 min (260 mM iodoacetamide in equilibration buffer). Equilibrated IPGs were transformed to 10% T, 2.5% C polyacrylamide gel without stacking gel; SDS-gel was run according to Laemmlie (1970). Two-dimensional gels were stained with silver according to the protocol of Swain and Ross (1995). RESULTS Extraction and recovery of proteins in recent and ancient bone The ﬁrst step in identifying proteins in recent and in archaeological bone samples is to establish the preservation state of organic and inorganic bone structures microscopically by viewing thin ground sections in plane and polarized light. In this way, INTACT BONE PROTEINS IN SKELETAL REMAINS 33 Fig. 1. Microstructure of well-preserved ancient compact bone substance. Thin ground section (50 m) viewed in polarized light. contamination and destruction caused by decomposition and diagenesis in ancient bone can be detected and ruled out (Hansen and Buikstra, 1987; Schoeninger et al., 1989; Schultz, 1986, 1997). As long as there is no contamination by plant roots, fungi, algae, bacteria, arthropods, and other contaminants, the bone can be used for protein extraction (Fig. 1). The dissociative demineralizing protocol used to prepare human bone samples is based on techniques developed since the beginning of the 1980s and adapted from vertebrate calciﬁed tissues (Termine et al., 1980). For this study, these techniques were modiﬁed. Guanidine hydrochloride, one of the strongest organic bases (pkb 0.3), was used to denature proteins. In combination with ethene diamine tetraacetic acid (EDTA), part of the calcium ions were chelated. The supernatant of the ﬁrst and the second extraction steps was removed, leaving only the tightly bound matrix proteins. A consequence of this procedure is the loosening of the apatite scaffold. This is a prerequisite for solubilization of strongly bound proteins of the extracellular bone matrix. When all calcium ions were chelated in our experiments, no proteins could be detected in gel electrophoresis. Bone proteins are precipitated with trichloracetic acid (TCA). Other concentration methods such as gel ﬁltration, ultraﬁltration, or dialysis cause a greater loss of bone NCPs. The silver-stained SDS-PAGE (Fig. 2) shows a distinctly resolved protein pattern, with molecular weights between 10 kD and about 150 kD. Figure 2 shows bone matrix proteins in SDS-PAGE after silver staining in recent freshly frozen and formalinﬁxed bones (Fig. 2A), and in ancient bones (Fig. 2B) dating from the late pre-Pottery Neolithic B (PPNB), through the Early Bronze Age (EBA), Iron Age (IA), and Early Middle Ages (EMA), to Early Modern Times (EMT). About 10 g protein can be solubilized from 20 mg BME. These matrix proteins are distinctly resolved in SDS-PAGE after silver staining to about 25 different bands. Fig. 2. Bone matrix proteins divided by SDS-PAGE (10% T, 2.5% C). A: Recent bone matrix proteins. Lane 1, frozen directly after dissection; lane 2, formalin ﬁxed for 1 month; lane 3, formalin ﬁxed for 1 year; lane 4, control collagen type 1; lane 5, molecular weight markers. B: Ancient bone matrix proteins. Lane 1, Gottorf (EMT); lane 2, Barbing (EMA); lane 3, Bajkara (IA/Scythian); lane 4, Franzhausen (EBA); lane 5, İkiztepe (EBA); lane 6, Basta (Late PPNB); lane 7, molecular weight markers. Intact collagenous and noncollagenous molecules in recent and ancient bones Different types of ECM human bone molecules were conﬁrmed with speciﬁc antibodies. Collagenous molecules (e.g., collagen type I, Fig. 3A), noncollagenous molecules (e.g., osteonectin, Fig. 3C), and molecules from the immune system (e.g., IgG, Figs. 3B, 5A) were identiﬁed by immunodetection. Collagen type I could only be conﬁrmed in freshly frozen recent bones and in some ancient bones (Bajkara, Kazakhstan; IA/Scythian; and Gottorf, Germany; EMT). The other samples of recent (formalin- 34 T.H. SCHMIDT-SCHULTZ AND M. SCHULTZ Fig. 4. 2-D separation from bone matrix proteins (Barbing, EMA): 23-g bone matrix proteins were loaded onto an IPG strip (pH 4 –7, from left to right). For second dimension, a vertical 10% SDS-gel was used, and proteins were visualized by silver stain. At right, molecular weight marker. Fig. 3. Western blot, with recent and ancient bone matrix proteins. A: Anti-human collagen type I. Lane 1, recent, freshly frozen; lane 2, ancient, Gottorf (EMT); lane 3, ancient, Bajkara (IA/Scythian); lane 4, human collagen type 1 control. B: Antihuman IgG. Lane 1, ancient, Barbing (EMA); lane 2, recent, freshly frozen; lane 3, ancient, Gottorf (EMT); lane 4, recent, formalin-ﬁxed for 1 month; lane 5, recent, formalin-ﬁxed for 1 year. Also see Figure 5a. C: Anti-human osteonectin. Ancient, Barbing (EMA). ﬁxed) and ancient bones gave no bands at all, or only weakly positive bands (Barbing, Germany; EMA) by immunodetection. In samples in which no collagen type I could be detected by antibodies, the noncollagenous protein pattern, however, was excellently expressed. These results demonstrate that the collagenous molecules are more susceptible to modiﬁcation or to the degradation process than noncollagenous molecules. Noncollagenous bone matrix molecules are apparently better protected by hydroxyapatite. ECM proteins of ancient bones separated by two-dimensional gel electrophoresis Here, ancient bone matrix proteins resolved by two-dimensional electrophoresis are demonstrated (Fig. 4). These techniques sort proteins according to two independent properties in two discrete steps: the ﬁrst-dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI). The second-dimension step (SDS-PAGE) separates proteins according to their molecular weight. Each spot on the two-dimensional array corresponds to a single protein in the sample. Twodimensional electrophoresis of the ancient bone from Barbing (21–25-year-old female) yields about 70 – 80 different spots. It is noteworthy that the protein content was only 23 g. The amount of proteins extracted from ancient bones is, as a rule, relatively low. Fig. 5. Check of IgG in ECM of bone of prehistoric children. A: Western blot of antibodies against the heavy chain of human IgG. Lane 1, Gemeinlebarn, 12–14 years old (EBA); lane 2, Franzhausen, 14 –16 years old (EBA); lane 3, İkiztepe, 8 –10 months old (EBA); lane 4, Bajkara, 12–13 years old (IA/Scythian); lane 5, İkiztepe, 2–3 years old (EBA). B: Thin ground section viewed in polarized light. Child no. 2, Franzhausen. Newly built bone formation (woven bone) at external surface of right humerus represents a hemorrhage characteristic of scurvy. Immunoglobulins in ancient bones A Western blot was performed with antibodies against the heavy chain of human IgG of 5 prehistoric children (Fig. 5A). The protein content of the samples was comparable (15 g). One of these 5 children (no. 2) suffered from scurvy, and it is well- INTACT BONE PROTEINS IN SKELETAL REMAINS known that chronic scurvy produces subperiosteal bleedings. The bone tissue can be changed by an organization process (healing) to a newly built woven-bone formation which frequently attracts macroscopic attention as bone apposition (e.g., Maat, 1982; Ortner, 2003; Schultz, 1993, 2001a). Only a microscopic investigation reveals a reliable diagnosis (Schultz, 2001b, 2003). Such changes caused by primary subperiosteal hematomas were observed on the surfaces of several long bones and the skull vault of child no. 2. Microscopic ﬁndings are presented in Figure 5B. This 14 –16-year-old child from Franzhausen has the lowest content of IgG. Thus, in this child, the chronic course of scurvy had resulted in a secondary immunodeﬁciency. DISCUSSION The most important research on detection of bone matrix proteins has been carried out in cell cultures (Ashton et al., 1985; Owen, 1988; Skjodt and Russel, 1992) and in grown bone tissue using histochemistry (Derkx et al., 1998; Kirsch and van der Mark, 1992; Tung et al., 1985). With increasing passages, these cell systems may lose their osteoblastic phenotype by dedifferentiation (Rao et al., 1977; Scott et al., 1980) or ﬁbroblast overgrowth (Rao et al., 1977). The results of in vitro studies obtained by various research groups working with osteoblast cell cultures are often not comparable with each other. Furthermore, immunohistochemistry cannot always prove the reliability of these results (Derkx et al., 1998; Tung, 1985), because this method is frequently prone to technical disadvantages. Samples taken for immunohistochemistry, whether from living bone tissue obtained by biopsy or from dry bone (e.g., forensic, anatomical, or archaeological collection materials), must be decalciﬁed. This decalciﬁcation, in addition to decomposition and diagenesis, might cause the structures of molecules to be changed so that antibodies can no longer recognize them (Amenta and Martinez-Hernandez, 1995; Derkx et al., 1998; Tung et al., 1985). This underlines the necessity to obtain results directly from the substrate bone, as shown in this paper. In contrast to other organic materials, bone tissue has a better chance of being preserved over a long time span due to its physical durability and resistance to decomposition and diagenesis. The conditions within the compact bone structure, which might also be characterized by the relatively low content of water and degradation enzyme, are favorable for the preservation of bone proteins. Extracellular matrix proteins of the living bone bind very tightly to hydroxyapatite. The apatite in which the proteins are embedded provides considerable protection against the destructive effects of temperature and chemical agents after death. In well-preserved bones, many thousands of years after death, the proteins of the extracellular matrix are conserved, as well as in fresh bones. With reliable techniques, it is possible to identify these proteins. 35 It is well-known that archaeological skeletal remains are affected in the earth by biogenic and abiogenic processes following burial, which together constitute the phenomenon of diagenesis (e.g., Hansen and Buikstra, 1987; Schoeninger et al., 1989; Schultz, 1986, 1997; Stout, 1987). Additionally, contamination occurring during and after archaeological excavation can alter the bone. These postmortem processes can falsify results of scientiﬁc examinations by affecting the identiﬁcation of trace elements, stable isotopes, glycoproteins (blood group factors), proteins, and DNA (cf. Schultz, 1997). Although attempts at a critical examination of such methods of investigation have been made, this complex subject, including the geochemistry of the burial environment (e.g., different soil conditions; ﬂow of water and soil crystals) and the wide scope of ﬂora and fauna (e.g., plant roots, bacteria, fungi, algae, and arthropods), has not been completely understood. However, the vestiges of these agents of diagenetic destruction are observable in the microscope, using plane and polarized light (e.g., Hackett, 1981; Schoeninger et al., 1989; Schultz, 1986, 1997, 2001b; Stout, 1987). Schoeninger et al. (1989, p. 291) noted that “it is possible to eliminate samples for analysis of trace elements in the inorganic fraction of bone when a thin section of the bone reveals a lack of histological structure.” This also holds true for the examination of proteins because of their tight binding to the bone apatite. Thus, for this study, only well-preserved samples were chosen. Most importantly, it is possible to extract proteins from ECM of ancient bone materials (ranging from PPNB to EMT) of the same quality as from recent bones. Up to now, there has been no presentation of such an elaborate, separated protein proﬁle extracted from ancient bone sample. Other groups have comparable results in recent human powdered bones (Fisher et al., 1987; Grynpas et al., 1994; Pope et al., 1980). The discovery of bone-protecting NCPs in ancient skeletal remains was ﬁrst mentioned in the late 1980s (Masters, 1987; Tuross, 1988, 1989; DeNiro and Weiner, 1988b), the results of which showed some very faint bands or more often a smear of NCPs after SDS-PAGE. This approach was followed, and the technique for solubilizing proteins of ECM was improved. The degreasing process of bone samples before powdering is vital for success (Waite et al., 1997). During the degreasing process, the protein pattern is not affected, and after the silver staining of the SDS-PAGE, the background is much clearer because of the elimination of lipids. Samples from various populations, located in different geographical areas and dating from different time periods, were selected. The intention was to demonstrate that not only a few samples taken from one population, but also many samples from different populations from different geographical areas and dating from different time periods, meet the conditions for extraction and identiﬁcation of ECM 36 T.H. SCHMIDT-SCHULTZ AND M. SCHULTZ proteins of ancient bones. Several examples of ancient bones which were poorly preserved, and showing various changes due to diagenesis, were also tested by the protein extraction procedure described in this paper. In these cases, the results showed no protein bands in the silver-stained extraction proﬁles. This may be explainable as follows. In the method described here, the supernatants of the ﬁrst and also the second protein extraction step were removed, because these supernatants contained all the proteins which were not tightly bound to the hydroxyapatite (danger of postmortem contamination), so that only the tightly bound proteins remained to be detected. During the intravitam aging process of bone, characteristic changes take place in the cortical and trabecular bone and also in the bone marrow. These changes are due to a variety of factors, including alterations in gonodal status, nutrition, and physical activity. The increase in active osteoclast numbers, with an increase in osteoclast life expectancy, results in uncoupling of bone turnover. This means that the total content of protein decreases; however, the protein pattern is the same, with only the distribution of NCPs being changed (Bergot et al., 1988; Chan and Duque, 2002; Parﬁtt et al., 1983). Thus, recent bone samples were used as a qualitative control of the protein pattern which is not a quantitative comparison. In Germany, recent samples taken from individuals of the biological age groups “subadultus” (0 –20 years old) and “adultus” (20 – 40 years old) are much more difﬁcult to obtain than from the biological age groups “maturus” (40 – 60 years old) and “senilis” (older than 60 years) because of legal conditions (e.g., donation of corpses during the lifetime of donors). Therefore, samples from the rare biological age groups “subadultus” and “adultus” are only used for direct quantitative comparison purposes. Collagen type I, osteonectin, and IgG were also conﬁrmed by other investigators in recent human bones (Bianco et al., 1988; Fisher et al., 1987; Grynpas et al., 1994; Pope et al., 1980; Trifﬁtt, 1987). However, in ancient human bone, intact collagen type I, osteonectin, and IgG molecules were shown to be present after thousands of years. Indeed, the quality of immunodetection in ancient bones was comparable to that in recent bones. Other research groups tried to isolate collagen type I from ancient bones (Collins and Galley, 1998; Semal and Orban, 1995; Tuross and Stathoplos, 1993). However, intact collagen type I chains in ancient bones in clear bands identiﬁed by antibodies are not reported in the literature. Some authors have voiced a suspicion that collagen type I molecules are degraded in ancient bones (Collins and Galley, 1998) or do not yield clear bands (Semal and Orban, 1995). Tuross et al. (1980) reported that proteins extracted by a 0.5-M EDTA demineralization and dialyses of bone powder yielded high molecular weight material that did not migrate. Tuross and Stathoplos (1993) showed some clearly separated protein bands with molecular weights in the range of osteonectin and IgG after SDS-PAGE. Formalin-ﬁxed powdered recent human bones were resolved by SDS-PAGE and, here also, a conserved matrix protein pattern between 10 –150 kD was found. Up to now, there are no comparable results on the protein pattern of powdered, formalinﬁxed recent bone in the literature. Formalin ﬁxed bones have sometimes been tested with histochemistry (Bosse et al., 1990). However, collagen type 1 could not be identiﬁed by speciﬁc antibodies in powdered, formalin-ﬁxed recent bones. Furthermore, only in some of the ancient bones (e.g., individuals from Gottorf, Bajkara, and Barbing) was collagen type 1 detected, although the pattern of the other matrix proteins found in other ancient specimens without positive proof of collagen type 1 (e.g., individuals from Basta, İkiztepe, Franzhausen, and Gemeinlebarn) was almost identical. One can assume that collagen molecules are more modiﬁed by various noxae (e.g., formalin) than the molecules of noncollagenous proteins. Thus, noncollagenous proteins are probably much better protected by bone apatite than are collagen molecules. There is much remaining work to do in identifying all the various proteins in bone. Intact molecules of collagen type 1, osteonectin, and IgG were shown to be detectable in principle, and the probability of identifying much more intact ECM proteins in ancient bones is very high. Experimental and observational studies (Bada et al., 1989; DeNiro and Weiner, 1988a; Weiner et al., 1989) indicate that perhaps several molecular-weight fractions result from the degradation of collagen. Using deﬁned laboratory conditions (such as short time periods in the range of minutes, known quantities of a speciﬁc protease, and incubation temperatures of, for instance, 25°C), the degradation process of bone collagen produces fragments larger than 10 kD (Peterkowsky, 1995). However, if not only one speciﬁc but a combination of many different speciﬁc proteases degrades bone tissue over a very long time (e.g., months, decades, hundreds, or even thousands of years), which is the case during diagenesis, the products of these degradation processes are very much smaller than 10 kD (Richards et al., 1993). Thus, these fragments cannot be detected correctly by SDS-PAGE because of their small size. In several publications, no patterns of bands were presented from SDS-PAGE because only smears (Tuross, 1978, 1988; Semal and Orban, 1995) were stained. Such a smear may contain many different structures such as apatite, lipids, and all proteins which are attached to them and which may be smaller or larger than 10 kD. These proteins, which as a rule are peptides, are not solubilized. This is a crucial condition, because exact separation of these proteins in SDS-PAGE and their detection by speciﬁc antibodies is not possible. The precondition for the exact separation and the identiﬁcation of bone INTACT BONE PROTEINS IN SKELETAL REMAINS matrix proteins using special antibodies is that all the proteins be solubilized (Merril and Washart, 1998). Proteome analysis in ancient bone Upon separation of proteins of the extracellular bone matrix using one-dimensional (1-D) electrophoresis, two or more different proteins with the same molecular weight may be located in the same protein band. However, using two-dimensional (2-D) electrophoresis, different proteins located in the same band can be easily differentiated. The bone matrix consists of about 200 different proteins (Boskey, 1992). If there is enough material (about 100 g protein) for 2-D electrophoresis, it must be possible to detect most if not all of the bone matrix proteins after silver staining in recent and also in well-preserved ancient bone. However, 2-D electrophoresis is very sensitive to interfering substances such as overly high salt concentrations. Therefore, extraction of extracellular matrix proteins should be carried out very carefully, using reliable techniques. Immunoglobulins in ECM of bone The extraction of intact IgG molecules from ancient bones opens up excellent possibilities. In this way, the history of infectious diseases may be more efﬁciently explored, e.g., the past history of reemerging human infections such as tuberculosis. The case of bovine spongiforme encephalopathy (BSE) underscores the point that DNA analyses cannot diagnose everything. Prions, the causative agents of BSE and scrapie, are especially durable proteins, which might persist for a long time period. Our ﬁrst intention was to ﬁnd IgG in samples of subadult skeletons because the immune system is, as a rule, highly stimulated in this age group. However, the skeletons of children are frequently not very well-preserved. Therefore, Bronze Age populations were chosen for research because, due to bronze grave goods, many parts of a skeleton may be preserved in a state similar to fresh bone. For comparative purposes, samples were taken not only from one but several populations. The bone samples taken from these Bronze Age children’s skeletons were in an excellent preservation state because of the protective agents of copper ions, which moved by diffusion from the bronze grave goods to the neighboring parts of the skeleton and stained not only the superﬁcial but also the interior bone tissue with a deep green color. Copper ions are bactericidal, and this is the reason for the excellent external and internal preservation of the bony tissue (cf. Schultz, 2001b). Additionally, the content of IgG in a bone sample of an Early Bronze Age child from Franzhausen (Austria) was very low. For the Western blot, 15 g protein per child were used. The protein assay for the quantitative precipitation (Bensadown and Weinstein, 1975) is very sensitive in the range of 1–50 g, because of the combined use of sodium 37 desoxychalate and trichloroacetic acid for precipitation. Additionally, this precipitation step separates the protein from interfering substances. The test and a standard curve in the range of 1–50 g bovine serum albumin (BSA) were used, and the results were checked several times. These ﬁndings correlate with the morphology of the microscopic investigation. Thus, the combination of protein-chemical (e.g., SDS-PAGE, 2-D gel electrophoresis) and morphological examinations (thin ground section analysis) results in exciting prospects for future work in this ﬁeld. Disease has always been a major determinant of life and death, whether past or present. In paleopathology, diseases are diagnosed macroscopically (Schultz, 1988b) and microscopically (Schultz, 1993, 2001b, 2003). These morphological investigations can be supported by the detection of speciﬁc antibodies preserved in ancient bones, yielding a successful combination of methods to make reliable diagnoses. PERSPECTIVES AND CONCLUSIONS There is now a chance to push forward with the proteomic of the ECM of bones from individuals from different time spans, from PPNB (and perhaps also earlier) to modern times. All biological molecules are potentially valuable for the study of the past; however, proteins and nucleic acids have the greatest potential. Hedges and Wallace (1978, p. 378) considered “that for example a comparison of several proteins (where they are available) could help determine the genetic relationship between Neanderthal and Anatomical Modern Man.” The wellknown morphological differences (Hublin et al., 1996; Schwartz and Tattersall, 1996) and new genetic results (Krings et al., 1997) tend to support a presumption that perhaps Neanderthals and modern humans descended from different human lines. Neither the genome (all DNA) nor the transcriptome (all RNAs) can reliably describe the physiology of cells and tissues. Proteins have undergone every biological process in the development, differentiation, and diseases of cells and tissues. Proteins are not the “underdogs;” they are the “real players” in cells and tissues. It could be a big step forward to extract intact extracellular matrix proteins from ancient bones with the same quality as from recent bones. The results of macroscopic and microscopic investigation, in combination with biochemical techniques such as 1-D and 2-D gel electrophoresis and Western blotting for the separation and identiﬁcation of the ECM protein pattern of an individual (or better, of several individuals of various populations from ancient times), open up a reliable opportunity to obtain much more information about the history and evolution of diseases. ACKNOWLEDGMENTS We gratefully acknowledge the generous support and stimulating advice given by Irmelin Probst (De- 38 T.H. SCHMIDT-SCHULTZ AND M. SCHULTZ partment of Biochemistry, University of Göttingen). For fresh bone samples, we thank E. Kunze, Department of Pathology, University of Göttingen (Germany), and C. Kuhnen, Department of Pathology, University of Bochum (Germany); for archaeological bone samples we thank Ö. Bilgi, Department of Ancient History, University of Istanbul, H.-G. Gebel, Department of Middle East Archaeology, University of Berlin, H. 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