NATURE AND DISTRIBUTION OF CHONDROITIN SULFATE AND DERMATAN SULFATE PROTEOGLYCANS IN RABBIT ALVEOLAR BONE

The type and distribution of mineral binding and collagenous matrix-as sociated chondroitin sulphate and dermatan sulphate proteoglycans in r abbit alveolar bone were studied biochemically and immunocytochemicall y, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The anti bodies specifically recognize oligosaccharide stubs that remain attach ed to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; cho ndroitin 6-sulphate and unsulphated chondroitin; and unsulphated chond roitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulph ate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fre sh specimens with a three-step extraction procedure: 4 M guanidine HCl (G-1 extract), 0.4 M EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix asso ciated proteoglycans in E- and G2-extracts, respectively Biochemical r esults using Western blot analysis of SDS-polyacrylamide gel electroph oresis of E- and G2-extracts demonstrated that mineral binding proteog lycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and der matan sulphate, whereas collagenous matrix associated proteoglycans sh owed a predominance of dermatan sulphate with a trace of chondroitin 4 -sulphate and no detectable chondroitin 6-sulphate or unsulphated chon droitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chond roitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were d istributed over the remainder of the bone matrix.