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The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth.

Coulson-Thomas YM, Coulson-Thomas VJ, Norton AL, Gesteira TF, Cavalheiro RP, Meneghetti MC, Martins JR, Dixon RA, Nader HB - PLoS ONE (2015)

Bottom Line: DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons.PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC.The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidade Federal de São Paulo, São Paulo, Brazil; School of Life Sciences, University of Lincoln, Lincoln, Lincs, United Kingdom.

ABSTRACT
Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

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Electrophoresis in PDA buffer of glycosaminoglycans extracted from archaeological bones and degraded with glycosaminoglycan-specific enzymes.GAGs were extracted from archaeological human adult and juvenile humerus (A), archaeological human adult and adolescent ulna (B and C), and archaeological human adult radius (D), subjected to β-elimination in some instances (samples 16 and 25), digested with chondroitin AC lyase, chondroitin ABC lyase, heparin lyase II or Flavobacterium heparinum extract, and analyzed by agarose gel electrophoresis in PDA buffer. The gels were stained with 0.1% toluidine blue prepared in a solution containing 1% acetic acid and 50% ethanol, destained with the same solution without toluidine blue, restained with 0.1% toluidine blue prepared in 25 mM sodium acetate buffer, pH 5.0, and destained in this solution without toluidine blue. CS: chondroitin sulfate; DS: dermatan sulfate; HS: heparan sulfate; Or: origin; St: standard (CS, DS and HS extracted from shark cartilage); C4S: chondroitin 4-sulfate; AC: chondroitin AC lyase; ABC: chondroitin ABC lyase; h’aseII: heparin lyase II; Flavo: Flavobacterium heparinum extract; 16: adult radius; 18: adult humerus; 19: juvenile humerus; 22: adolescent ulna; 24: adult ulna; 25: adult ulna.
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pone.0131105.g002: Electrophoresis in PDA buffer of glycosaminoglycans extracted from archaeological bones and degraded with glycosaminoglycan-specific enzymes.GAGs were extracted from archaeological human adult and juvenile humerus (A), archaeological human adult and adolescent ulna (B and C), and archaeological human adult radius (D), subjected to β-elimination in some instances (samples 16 and 25), digested with chondroitin AC lyase, chondroitin ABC lyase, heparin lyase II or Flavobacterium heparinum extract, and analyzed by agarose gel electrophoresis in PDA buffer. The gels were stained with 0.1% toluidine blue prepared in a solution containing 1% acetic acid and 50% ethanol, destained with the same solution without toluidine blue, restained with 0.1% toluidine blue prepared in 25 mM sodium acetate buffer, pH 5.0, and destained in this solution without toluidine blue. CS: chondroitin sulfate; DS: dermatan sulfate; HS: heparan sulfate; Or: origin; St: standard (CS, DS and HS extracted from shark cartilage); C4S: chondroitin 4-sulfate; AC: chondroitin AC lyase; ABC: chondroitin ABC lyase; h’aseII: heparin lyase II; Flavo: Flavobacterium heparinum extract; 16: adult radius; 18: adult humerus; 19: juvenile humerus; 22: adolescent ulna; 24: adult ulna; 25: adult ulna.

Mentions: GAGs were extracted from archaeological human bones and teeth and analyzed by agarose gel electrophoresis in PDA buffer, whereby GAGs are separated according to their affinity with the buffer and can be visualized by toluidine blue staining co-migrating with the standard control (composed of CS, DS and HS). GAGs extracted from a modern wisdom tooth served as a comparative positive control. The electrophoretic profile of the GAGs isolated from bone and tooth samples revealed the presence of a CS population for all samples, detected as a single band in the gels (Fig 1A and 1C). This CS population was confirmed to be CS chains by treatment with specific glycosidases as discussed below. When the same agarose gels were restained and destained with sodium acetate buffer, HA was also revealed for all samples, detected as a streak in the gels (Fig 1B and 1D). The GAG profiles were similar for all samples; however, a third GAG population that co-migrated with HS was also detected in two samples; adult radius and adult ulna (arrows in Fig 2).


The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth.

Coulson-Thomas YM, Coulson-Thomas VJ, Norton AL, Gesteira TF, Cavalheiro RP, Meneghetti MC, Martins JR, Dixon RA, Nader HB - PLoS ONE (2015)

Electrophoresis in PDA buffer of glycosaminoglycans extracted from archaeological bones and degraded with glycosaminoglycan-specific enzymes.GAGs were extracted from archaeological human adult and juvenile humerus (A), archaeological human adult and adolescent ulna (B and C), and archaeological human adult radius (D), subjected to β-elimination in some instances (samples 16 and 25), digested with chondroitin AC lyase, chondroitin ABC lyase, heparin lyase II or Flavobacterium heparinum extract, and analyzed by agarose gel electrophoresis in PDA buffer. The gels were stained with 0.1% toluidine blue prepared in a solution containing 1% acetic acid and 50% ethanol, destained with the same solution without toluidine blue, restained with 0.1% toluidine blue prepared in 25 mM sodium acetate buffer, pH 5.0, and destained in this solution without toluidine blue. CS: chondroitin sulfate; DS: dermatan sulfate; HS: heparan sulfate; Or: origin; St: standard (CS, DS and HS extracted from shark cartilage); C4S: chondroitin 4-sulfate; AC: chondroitin AC lyase; ABC: chondroitin ABC lyase; h’aseII: heparin lyase II; Flavo: Flavobacterium heparinum extract; 16: adult radius; 18: adult humerus; 19: juvenile humerus; 22: adolescent ulna; 24: adult ulna; 25: adult ulna.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4481269&req=5

pone.0131105.g002: Electrophoresis in PDA buffer of glycosaminoglycans extracted from archaeological bones and degraded with glycosaminoglycan-specific enzymes.GAGs were extracted from archaeological human adult and juvenile humerus (A), archaeological human adult and adolescent ulna (B and C), and archaeological human adult radius (D), subjected to β-elimination in some instances (samples 16 and 25), digested with chondroitin AC lyase, chondroitin ABC lyase, heparin lyase II or Flavobacterium heparinum extract, and analyzed by agarose gel electrophoresis in PDA buffer. The gels were stained with 0.1% toluidine blue prepared in a solution containing 1% acetic acid and 50% ethanol, destained with the same solution without toluidine blue, restained with 0.1% toluidine blue prepared in 25 mM sodium acetate buffer, pH 5.0, and destained in this solution without toluidine blue. CS: chondroitin sulfate; DS: dermatan sulfate; HS: heparan sulfate; Or: origin; St: standard (CS, DS and HS extracted from shark cartilage); C4S: chondroitin 4-sulfate; AC: chondroitin AC lyase; ABC: chondroitin ABC lyase; h’aseII: heparin lyase II; Flavo: Flavobacterium heparinum extract; 16: adult radius; 18: adult humerus; 19: juvenile humerus; 22: adolescent ulna; 24: adult ulna; 25: adult ulna.
Mentions: GAGs were extracted from archaeological human bones and teeth and analyzed by agarose gel electrophoresis in PDA buffer, whereby GAGs are separated according to their affinity with the buffer and can be visualized by toluidine blue staining co-migrating with the standard control (composed of CS, DS and HS). GAGs extracted from a modern wisdom tooth served as a comparative positive control. The electrophoretic profile of the GAGs isolated from bone and tooth samples revealed the presence of a CS population for all samples, detected as a single band in the gels (Fig 1A and 1C). This CS population was confirmed to be CS chains by treatment with specific glycosidases as discussed below. When the same agarose gels were restained and destained with sodium acetate buffer, HA was also revealed for all samples, detected as a streak in the gels (Fig 1B and 1D). The GAG profiles were similar for all samples; however, a third GAG population that co-migrated with HS was also detected in two samples; adult radius and adult ulna (arrows in Fig 2).

Bottom Line: DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons.PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC.The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidade Federal de São Paulo, São Paulo, Brazil; School of Life Sciences, University of Lincoln, Lincoln, Lincs, United Kingdom.

ABSTRACT
Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

No MeSH data available.


Related in: MedlinePlus