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

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 skeleto ns. 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

Immunolocalization of chondroitin-6-sulfate in archaeological human bone slices.

<p>Archaeological human adult humerus or femur slices (60 μm thick) were labeled with anti-chondroitin-6-sulfate (following digestion with chondroitin AC lyase) and developed using DAB (brown). The secondary control was prepared omitting the primary antibody (archaeological human adult femur is shown). Cortical bone is viewed on the left and trabecular bone on the right. Scale bar: 200 μm.</p

Immunolocalization of biglycan in archaeological human juvenile and adolescent bone slices.

<p>Archaeological human juvenile humerus and adolescent femur slices (70 μm thick) were labeled for biglycan and developed using DAB (brown). Cortical bone is viewed on the left and trabecular bone on the right. Scale bar: 200 μm.</p

Proteoglycans extracted from ancient bones and teeth.

<p>PGs were extracted from various types of archaeological human bone samples (femur, humerus, radius, tibia and ulna) identified as juvenile, adolescent or adult, and also archaeological adult teeth and a modern wisdom tooth. Extracted PGs were analyzed by agarose gel electrophoresis in PDA buffer, and stained by amide black followed by toluidine blue. Proteins are stained in blue and GAG chains in purple. CS: chondroitin sulfate; DS: dermatan sulfate; HS: heparan sulfate; St: standard (CS, DS and HS extracted from shark cartilage).</p

Disaccharide content of the CS population extracted from archaeological human adolescent femur was characterized by strong anion exchange liquid chromatography.

<p>(A) C6S standard profile; (B) C4S/C6S Standard profile; and (C) profile of CS extracted from archaeological human adolescent femur. (a) Peak representing the C6S disaccharide; and (b) peak representing the C4S disaccharide.</p

Immunolocalization of BSPII, osteocalcin and collagen I in archaeological human bone slices.

<p>Archaeological human femur slices (60 μm thick) were labeled for BSPII, osteocalcin and collagen I, and developed using DAB (brown). Cortical bone is viewed on the left and trabecular bone on the right. Scale bar: 200 μm.</p