Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone.
We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.
Takagi
M
M
Department of Anatomy, Nihon University School of Dentistry, Tokyo, Japan.
Maeno
M
M
Kagami
A
A
Takahashi
Y
Y
Otsuka
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001842:Bone and Bones / Q000737:chemistry* / Q000648:ultrastructure; D002807:Chondroitin / Q000032:analysis; D002808:Chondroitin Lyases / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000032:analysis; D003871:Dermatan Sulfate / Q000032:analysis; D004492:Edetic Acid; D019791:Guanidine; D006146:Guanidines; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008903:Minerals / Q000378:metabolism*; D010011:Osteocytes / Q000737:chemistry / Q000648:ultrastructure; D011509:Proteoglycans / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D006146:Guanidines; D008903:Minerals; D011509:Proteoglycans; D003871:Dermatan Sulfate; D002807:Chondroitin; D002809:Chondroitin Sulfates; D004492:Edetic Acid; D002808:Chondroitin Lyases; D019791:Guanidine