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Bone protects proteins over thousands of years Extraction analysis and interpretation of extracellular matrix proteins in archeological skeletal remains.

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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: mschult1@gwdg.de
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. Parzinger, German Archaeological Institute (DAI), Berlin (Germany), P. Schröter, Anthropological State Collection, Munich (Germany),
M. Teschler-Nicola, Museum of Natural History, Vienna (Austria), and H. Wolf, Department of Medical
History, University of Kiel (Germany). Furthermore, we thank M. Brandt, S. Dessi, and C.
Maelicke (all at the Department of Anatomy, University of Göttingen) for technical assistance. Additionally, the authors particularly thank the reviewers who helped in finalizing the text of this
contribution.
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