AbstractsBiology & Animal Science

Characterization of Non-collagenous Proteins in Vertebrate Mineralization

by Ling Chen




Institution: University of Akron
Department: Polymer Science
Degree: PhD
Year: 2014
Keywords: Polymers; Biology
Record ID: 2028044
Full text PDF: http://rave.ohiolink.edu/etdc/view?acc_num=akron1405950378


Abstract

Mineralization of vertebrate tissues such as bone, dentin, cementum, and calcifying tendon involves type I collagen, which has been proposed as a template for calcium and phosphate ion binding and subsequent nucleation of apatite crystals. Type I collagen thereby has been suggested to be responsible for the deposition of apatite mineral without the need for non-collagenous proteins or other extracellular matrix molecules. Based on studies in vitro, non-collagenous proteins, including osteocalcin and bone sialoprotein, are thought to mediate vertebrate mineralization associated with type I collagen. These proteins, as possibly related to mineral deposition, have not been definitively localized in vivo. The present study has reexamined their localization in the leg tendons of turkeys, a representative model of vertebrate mineralization in certain avian species. Immunocytochemistry of osteocalcin demonstrates its presence at the surface of, outside or within type I collagen while that of bone sialoprotein appears localized at the surface of or outside type I collagen. The association between osteocalcin and type I collagen structure is revealed optimally when calcium ions are added to the antibody solution in the immunocytochemistry methodology. In this manner, osteocalcin is found specifically located along the a<sub>4-1</sub>, b<sub>1</sub>, c<sub>2</sub> and d bands defining in part the hole and overlap zones within type I collagen. From these data, while type I collagen itself may be considered a stereochemical guide for intrafibrillar mineral nucleation and subsequent deposition, osteocalcin bound to type I collagen may also possibly mediate nucleation, growth and development of platelet-shaped apatite crystals. Bone sialoprotein and osteocalcin as well, each immunolocalized at the surface of or outside type I collagen, may affect mineral deposition in these portions of the avian tendon.Calcification of the tendon occurs in a series of spatial-temporal events associated with gene expression, protein synthesis and secretion, and structural changes in this tissue. Non-collagenous proteins and other molecules mediating mineralization are expected to be expressed, synthesized and secreted in a spatial-temporal manner similar to that of calcification. The present study has attempted to correlate the onset and progression of calcification in turkey leg tendons with the gene expression of several extracellular matrix molecules in this tissue using reverse transcription-quantitative polymerase chain reaction analysis (RT-qPCR). The molecules examined included osteocalcin, bone sialoprotein, osteopontin, vimentin, and decorin. It was found that the mRNA expression of bone sialoprotein, osteopontin, and osteocalcin corresponds well with the mineralization process. Immunolocalization separately confirms the synthesis and secretion of these matrix molecules. However, the expression of other non-collagenous molecules such as decorin does not show strong correlation with turkey leg tendon mineralization. Proteolytic degradation…