1997 Fiscal Year Final Research Report Summary
Keratan sulphate proteoglycans associated with the bone formation of embryonic chick femurs
| Project/Area Number |
08672099
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Morphological basic dentistry
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| Research Institution | Nihon University |
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Principal Investigator |
TAKAGI Minoru Nihon University School of Dentistry Professor, 歯学部, 教授 (90060061)
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| Co-Investigator(Kenkyū-buntansha) |
SHIGEMASA Kayoko Nihon University School of Dentistry Research Associate, 歯学部, 助手 (30059604)
MAENO Masao Nihon University School of Dentistry Associate Professor, 歯学部, 助教授 (60147618)
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| Project Period (FY) |
1996 – 1997
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| Keywords | bone / chick embryo / glycosaminoglycan / proteoglycan / keratan sulphate / immunohistochemistry / Western blot |
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| Research Abstract |
The type and sistribution of sulphated proteoglycans (PGs) in the midshaft subperiosteal bone of 15-18-day embryonic chick femurs were studied immunocytochemically and biochemically, using four monoclonal antibodies (MAb 2B6, 3B3, 1B5, and 5D4). These MAb specifically recognize epitopes in chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS) ; chondroitin 6-sulphate (C6-S) and unsulphated chondroitin (C0-S) ; C0-S ; and keratan sulphate (KS) respectively. Immunohistochemistry showed that staining of C4-S, DS, and KS, but not of C6-S and C0-S, was limited to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunae and bone canaliculi in 15-18-day embryonic specimens. However, no significant difference in the distribution and intensity of immunostaining was observed in these specimens. Bone proteins were extracted from fresh 18-day embryonic specimens with a three extraction procedure, 4 M guanidine HCl (GdnCl, G-1 extract), 0.4M EDTA (E-ectract), followed by GdnCl (G-2extract), to characterize mineral binding and collagenous matrix associated PGs in E-and G2-extracts respectively. Western blot analysis of E-and G2-extracts demonstrated that chondroitinase ABC-digested PGs with a molecular weight (M_r) approximately of 45,000 containing GAGs predominantly corresponding to C4-S and/or DS, with no detectable C6-S or C0-S present in the mineral and matrix phase, whereas KSPGs having an M_r of approximately 72,000 are only present in the mineral phase. These results indicate that embryonic chick bone contains small PGs having C4-S, DS, and KS chains with preferential localization to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunac and bone canaliculi.
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