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Connective Tissue Research, 44(Suppl. 1): 41–46, 2003
Copyright
c
°
2003 Taylor & Francis
0300-8207/03 $12.00 + .00
DOI: 10.1080/03008200390152070
Identification of Proteinaceous Material in the Bone
of the Dinosaur
Iguanodon
Graham Embery,
1
Angela C. Milner,
2
Rachel J. Waddington,
3
Rachel C. Hall,
1
Martin S. Langley,
3
and Anna M. Milan
1
1
Department of Clinical Dental Sciences, University of Liverpool, Liverpool, United Kingdom
2
Department of Palaeontology, Natural History Museum, London, United Kingdom
3
Department of Basic Dental Science, University of Wales College of Medicine, Cardiff, United Kingdom
This study has directed attention at the search for bone-related
proteins in an extract of demineralized rib bone of the 120 mya
Iguanodon. The inner compact bone was demineralized and the
GuCl extractresolved into11 fractions using anion exchange chro-
matography, which all contained silver-reactive proteins with var-
ious amino acid profiles. Two specific fractions, iv and xi, revealed
characteristics typical of contemporary phosphoproteins and pro-
teoglycans, respectively. Fraction iv, 43–57 kDa, contained a high
ratio of aspartate and serine, although no phosphate was discern-
able. Fraction xi contained a band of 41–47 kDa and was rich in
chondroitin sulphate and hyaluronan. In addition an early eluting
fraction was immunoreactive with an antibody against osteocalcin.
A cancellous bone fraction from the same bone sample was also
analyzed using N-terminal sequencing and revealed potential sim-
ilarities with cystatin. While we do not claim to have identified the
presence of intact proteins, this study has value in demonstrating
that extruded extracellular matrix is protected by its capacity to
inducemineralization,whichsubsequentlyisimportantinconserv-
ing detectable protein products in ancient skeletal tissues.
Keywords Bone, Dinosaur, Iguanodon, Protein.
INTRODUCTION
The potential for preservation of ancient biomolecules has
beendemonstratedincreasinglyclearlyinrecentyears[1].Many
studies have been directed toward the search for DNA in both
animal and plant material. DNA sequences have been recovered
from a variety of subfossils [2, 3] and have been reported from
much older animal material. Fragmentary DNA was reported
from Late Jurassic dinosaur bones from a coal mine in Utah [4]
Received 9 November 2001; revised 2 February 2002; accepted 2
March 2002.
Address correspondence to Professor G. Embery, Department of
Clinical Dental Sciences, Edwards Building, University of Liverpool,
Liverpool, UK, L69 3GN. E-mail: g.embery@liv.ac.uk
but was later shown to be a mix of bacterial, fungal, and hu-
man contaminants [5, 6]. DNA also was reported from the Late
Cretaceous tyrannosaurid Tarbosaurus, on the basis of histo-
chemical studies on isolated osteocytes [7], although this work
has not been followed up or independently repeated. Claims of
isolation of authentic DNA from amber-preserved insects and
plant inclusions and other ancient material remain controver-
sial, largely because they have not been verified by independent
replication,a primary criterionof authenticity [8].AncientDNA
sequencesfromspecimensyoungerthan100,000yearshavenow
been replicated independently, but work on older material has
not been reproducible [8].
Proteins and polysaccharides associated with vertebrate hard
tissues have the most obvious potential for preservation over
long-duration geologic time. Fossilization implies a process
wherebythe hard tissuesofan organismarecompletely replaced
withinorganicminerals,preserving structure ratherthanorganic
components. However, fossils exhibit varying states of preser-
vation from very little alteration to complete mineral replace-
ment [9]. Incorporation of bone proteins into the microcrys-
talline structure appears to be an important factor in facilitating
their long-term preservation.
Histochemicaldetection ofmucopolysaccharides in dinosaur
bone was first reported in 1972 [10]. Collagen has been isolated
from fossil bird, mammal, and reptile bone including dinosaurs
[11]. Proteins, particularly those associated with collagen, have
been recovered from the Upper Jurassic sauropod dinosaur Seis-
mosaurus; the relative abundance of amino acids also suggested
the presence of proteins other than collagen [12]. High levels of
proline, glycine, and hydroxylysine in an unusually nonpermin-
eralized, mumified individual of the Late Cretaceous theropod
Tyrannosaurus furnished unusually complete evidence of colla-
gen preservation in 65 million year old bone [11].
Although immunological research of fossil bone proteins has
focused on collagen [13, 14], the noncollagenous bone protein,
41
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42 G. EMBERY ET AL.
osteocalcin, has been detected in a wide range of subfossil and
fossil bird, mammal and reptile material [15]. The presence of
osteocalcin in dinosaur bone from Upper Cretaceous ceratopi-
ans (horned dinosaurs), hadrosaurs (duck-billed dinosaurs), and
an Upper Jurassic sauropod was detected immunologically and
verified by reverse-phase HPLC [15]. Osteocalcin is strongly
bound to the bone mineral and its preservation in fossil bone
appears to depend more on diagenesis and taphonomy and not
simply on the age of the material [15]. Following a challenge
that the kinetics of protein decomposition are inconsistent with
its long-term preservation, studies of artificial aging of modern
bone samples have demonstrated that preservation of osteocal-
cin cannot be excluded on kinetic grounds at ambient burial
temperatures of 10
◦
C and confirmed the importance of mineral
association to protein survival [16].
In an extension of this reasoning, studies have also extended
into the types of noncollagenous proteins likely to be retained
within fossilized mineralized bone. The identification of such
material would be of particular benefit in ascribing these mole-
culesrolesinepitacticalnucleationtogetherwiththeiridentifica-
tion as bone-related proteins in comparison with contemporary
material. The investigation now reported seeks to identify and
further characterize bone proteins in the bone of the dinosaur
Iguanodon. The work presented within this article extends the
work of Embery et al. [17] including more detailed analysis of
specific compact bone fractions and N-terminal sequencing of a
fraction from the closely associated cancellous bone.
MATERIALS AND METHODS
Methods for the preparation of the bone samples, deminer-
alization and anion-exchange chromatography were essentially
those used by Embery et al. [17]. The bone sample was taken
fromawell-preservedriboftheornithopoddinosaurIguanodon.
It was an authenticated specimen in the collection of the Nat-
ural History Museum of London (UK) and was 125–135 mya.
The extraction process involved sectioning of the rib into 10-cm
portions, and the compact bone was separated by carefully chis-
ellingthesamplesinlongitudinalsection.Thisyieldedlongitudi-
nalplate-likebonesampleswitha defined Haversianstructureas
evidenced by light microscopy. Throughout the extraction pro-
cedure, every effort was made to reduce contamination and han-
dling. Principal analyses were performed on the inner compact
bone, 1 cm in depth from the central cortex, with outer portions
being discarded. Samples of cancellous bone were additionally
analyzed from the marrow region and used for N-terminal se-
quencing because insufficient material remained from the corti-
cal bone for further biochemical characterization.
These inner bone samples were crushed to a fine powder and
demineralized in 10% EDTA, pH 7.4 for 7 days at 4
◦
C, followed
byfurther extractionofthe organicmatrixwith 4M guanidinium
chloride (GuCl) in 0.05M sodium acetate at pH 5.8 for 3 days
[17]. Following dialysis and lyophilization, the organic matrix
extract was separated by anion exchange chromatography, in
7M urea, 0.05M Tris-HCl pH 6.5 with a stepped gradient of
0–2 M NaCl selectively eluting matrix components. These frac-
tions were pooled accordingly, dialyzed, lyophilized and sub-
jected to SDS-PAGEonthe Phastsystem(Amersham Pharmacia
Biotech) as described by Embery et al. [17]. Of note, equal pro-
tein concentrations were loaded onto the gel for each fraction.
Amino acid analysis was also performed following acid hydrol-
ysis and separation on a Pickering cation exchange amino acid
analysis column integrated into a Dionex HPLC system. Im-
munologicalidentificationof boneextractswasalsoinvestigated
byprobing aliquotsof thefractions of interest blotted onto nitro-
cellulose with antibodies against osteocalcin, bone sialoprotein,
and osteopontin as described previously [17]. In addition cel-
lulose acetate electrophoresis to determine the presence of gly-
cosaminoglycans before and after digestion with protease-free
chondroitinase ABC also was examined. N-terminal sequenc-
ing of fraction iv from the cancellous bone elution profile was
determined by Edman degradation, where the fraction was blot-
ted onto PVDF membrane, and the N-terminal sequence deter-
mined by sequentially removing amino acid residues from the
N-terminus of the protein and identifying them by reverse-phase
HPLC.Dr.P.Neame,University ofSouthFlorida,whose analyt-
ical expertise is acknowledged here, performed the sequencing.
RESULTS
Extraction was performed on both the bone marrow or can-
cellous bone and the compact bone, dissected as described in
the Methods section. Following EDTA demineralization and
GuCl extraction, an organic matrix extract was obtained from
bothsources.TheseEDTA/GuClextractswere readilysolublein
urea and were subsequently separated by anion exchange chro-
matography to yield the profile in Figure 1. Several peaks were
witnessed from both sources, by absorbance at 280 nm, with
more material present within the compact bone samples. The
compact bone elution profile was resolved into the fractions as
detailed on the elution profile. A previous study has identified
proteinaceous material in all these fractions [17], and the SDS-
PAGEseparationsand amino acidanalyses ofthese fractions are
Displayed in Figure 2. However, this study focussed primarily
on extending the biochemical characterization of two fractions
from the compact bone profile, fraction iv and fraction xi.
Fraction IV
Following anion exchange chromatography (Figure 1), frac-
tion iv eluted between 0.3–0.42M NaCl and contained a broad
band between 43–57 kDa as determined by SDS-PAGE
(Figure 2). The amino acid profile of this fraction indicated a
high ratio of aspartate, serine, and glycine residues (Figure 2),
with aspartate and serine accounting for 52.8% of the total
residues.Comparison can bemade with contemporaryphospho-
proteins that are rich in aspartate and serine residues [18–22].
However, phosphate analysis of this factionfailed to identify the
presence of phosphate groups following hydrolysis of this frac-
tion. Previous reports have indicated that these protein bands
are probably fragments of the original protein; however, it is
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PROTEINS OF DINOSAUR BONE 43
Figure 1. Elution profile of the GuCl extract from a Resource-Q anion exchange chromatography column integrated into a FPLC system using a stepped 0–2M
NaCl gradient. Key:———— = compact bone;----=cancellous bone;—-—-—=NaCl gradient.
possible to perform a comparison of the amino acid ratios with
knownproteins using the ExPASy AACompIdent-Constellation
program. Care must be taken in interpretation as these proteins
will not be directly represented within the database. However,
this program has suggested several potential comparisons, the
top three of which are a eukaryotic prostatic spermine-binding
protein that is a glycoprotein involved in binding polyamines;
a eukaryotic calcium-binding acidic rich repeat protein; a pre-
cursor to calfluxin that is involved in influx of calcium unto the
mitochondria.
These results corroborate that this fraction is a glycoprotein
that potentially had the capacity for post-translational modifica-
tions including glycosylation and phosphorylation.
Fraction XI
The second fraction of interest was fraction xi. Following an-
ion exchange chromatography this fraction eluted when a max-
imum NaCl gradient of 2M was applied to the column, indicat-
ing material highly anionic in nature. Molecular weight analysis
identified a band of 41–47 kDa, which upon comparison with
contemporary proteins has a characteristic molecular weight
of small-leucine-rich proteoglycan core protein [23]. Cellulose
acetate electrophoresis (Figure 3) revealed that this fraction
containedchondroitin sulphate,a predominant glycosaminogly-
can of mineralized tissues, and hyaluronan. Calculations upon
comparisonwithknownglycosaminoglycanstandardsidentified
that fraction xi was composed of 48.4 ± 2.8% chondroitin sul-
phate and 51.6 ± 2.7% hyaluronic acid. Following chondroiti-
nase ABC digestion of fraction xi, the band was seen to disap-
pear (data not shown)confirming identification of glycosamino-
glycans. Amino acid analysis of this fraction revealed greater
than average number of leucine residues, with 40 per 1000
determined, which is a characteristic feature of these proteins
isolated from dentine and bone [24]. In addition, comparing
the amino acid ratios with known proteins using the ExPASy
AACompIdent-Constellation program suggested similarities to
aggrecan core protein, with matches in most species including
canine, bovine, rat, and human. Preliminary characterization of
fraction ii was also performed, with positive immunoidentifi-
cation of osteocalcin using a mouse monoclonal antibody. A
slight positive result was also identified in fraction i. However,
no other fractions were immunoreactive to osteocalcin antibod-
ies. Other antibodies tested represented polyclonal antibodies
raised in rabbit, in all these cases the secondary antirabbit an-
tibody was reactive to all proteins extracted from the dinosaur
compact bone.
Due to the amount of material obtained from the cancel-
lous bone fraction of the Iguanodon, as a preliminary charac-
terization, fraction iv from the elution profile (Figure 1) was
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Figure 2. Silver stained SDS-PAGE electrophoretic separations of compact bone fractions i–xi (equal protein concentration loaded 10 mg/ml) and their corresponding amino acid profiles. Amino acid
profiles are shown as residues per 1000 total residues.
44
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PROTEINS OF DINOSAUR BONE 45
Figure 3. Cellulose acetate electrophoresis of fraction xi stained with alcian blue indicating the presence of glycosaminoglycan components prior to protease
free chondroitinase ABC digestion. Following digestion, alcian blue stained material was not apparent.
subjected to N-terminal sequencing to determine any partial
or fragmented protein present. The determined sequence was
KXLPGTNEDLVLXG, and comparison using a BLASTsearch
[25] against a mammalian database including human, revealed
many potential comparisons, the predominant ones of which
follow:
Iguanodon K X L P G T N E D L V L X G
Cystatin 71 K S LPG
Q
NEDLVLTG 84
Gastrin precursor 33 S S G PGTNEDLEQRQ 46
Annexin 1 G W GTNEDLVISI 12
Yes associated protein 415 E A LPGTNVDLGTLE428
Chondroitin sulphate 931PVT S FTNEDLI H G R 944
proteoglycan NG2
Laminin 488 R D T R L S A E DLVLEG501
Aggrecan I 451PATAFTSEDLVVGV465
While cystatin has the highest number of matching amino
acids, it is important to remember that this fraction is likely to
be composed of a fragmented protein, so sequence comparison
further along protein sequences are also likely, allowing poten-
tial matches with several other matrix proteins, containing the
sequence motif TNEDL.
DISCUSSION
The results presented here confirm and extend our previous
observations [17] on some noncollagenous components of the
compact bone of the dinosaur Iguanodon and introduces some
early work on cancellous (marrow) bone from the same sample.
A range of protein-containing fractions from both bone samples
were obtained by anion exchange chromatography using FPLC,
following EDTA demineralization and GuCl extraction, with a
range of proteins identified using SDS-PAGE.
In this study particular attention has been directed at fraction
ivand xi from the compact bone and fraction iv from the cancel-
lous bone, the latter being used for N-terminal sequencing. The
aminoacidprofiles of fractioniv indicated thetentativepresence
of phosphoproteins. Such proteins are evident in bone and den-
tinesamplesof contemporary sources andofnoteisthe presence
of a 60 kDa molecular weight species in an avian sample [26].
A similar molecular weight species could be α
2
-HS glycopro-
tein, which is also rich in aspartate, glutamate, and alanine. The
protein is of liver origin, a member of the cystatin super gene
family, and is sequestered in mineralized tissues of higher
orders.
ThefractionivofcancellousbonewassubjectedtoN-terminal
sequencing. The 55 kDa protein that probably represents a frag-
mented protein as a feature of time or by free radical degrada-
tion, was able to display sufficient protein sequence assemblies
to enable comparison with protein databases and suggest poten-
tial comparisons with sequences found in cystatin, annexin, and
gastrin precursor. The N-terminal sequencing data are our best
available evidence since the study is based on limited quantities
of starting material, which in itself is 120–130 mya.
Fraction xi from the compact bone extract was a leucine-rich
fraction by comparison to the amino acid profiles of the other
fractions and eluted when a maximum molarity of 2M was ap-
plied to the anion exchange column, a feature of leucine-rich
proteoglycans. The fraction contained chondroitin sulphate, a
classical component of mineralized tissues in mammalians and
higher orders of the animal kingdom, and its presence also cor-
roborates the earlier histochemical evidence on the presence
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46 G. EMBERY ET AL.
of mucopolysaccharides [10]. The detection of chondroitin-
sulphate containing protein in this fraction is in keeping with
a proposed role in potentiating, along with type I collagen and
otherextracellularmatrix factors,epitacticcrystal growth.Simi-
lar compositional arrangements would be found in modern bone
samples and represent the synthesis and conservation at an early
geological time of a uniquely successful assembly for mineral-
ized tissues.
Early eluting fractions from the compact bone also revealed
immunoreactivity toward osteocalcin. This glycoprotein has
been found in a number of ancient tissue samples and together
withthe tentativepresence ofa phosphoprotein, chondroitin sul-
phate proteoglycan, and possibly cystatin indicate that we have
detected a variety of bone-specific or bone-characteristic pro-
teins. This lends credibility to our claim that we have isolated
and detected authentic proteins from fossil bone.
Such findings overcome previous arguments levelled against
claims to have detected DNA from such material, the suscep-
tibility to change of DNA over minimum time periods and its
ubiquitous presence in all species. A further aspect is the in-
tracellular nature of nucleic acid material, which could well be
lost as a result of cellular activity in the degenerative phase.
By comparison, the extracellular matrix is an extruded material,
which is possibly protected by its capacity to induce mineral-
ization and therefore has a much higher possibility of being
protected relatively unchanged over long periods of time. We
contend that this is a formative outcome of this and previous
studies.
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