PURPOSE: To develop a novel means of conjugating bisphosphonates onto the carbohydrate moieties of glycoproteins to enhance protein affinity to bone. METHODS: 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the carbohydrate moietites of oxidized fetuin by using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (MMCCH). Bone affinity of the resulting conjugates was compared to proteins obtained from another means of conjugation, whereby aminoBP was conjugated onto fetuin's lysine moieties by using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). RESULTS: The use of the MMCCH resulted in the conjugation of up to seven aminoBPs per molecule of fetuin. These conjugates gave a 2.6-, 2.0-, 30.5-. and 1.84-fold increased affinity for untreated, ashed, demineralized bone and hydroxyapatite, respectively, as compared to conjugates from the SMCC reaction. Both conjugates exhibited a pH-independent, equally slow degradation in adult bovine serum-containing media. CONCLUSION: The use of the MMCCH chemistry to conjugate aminoBP onto fetuin was feasible. Furthermore, the described processes of conjugation resulted in amino-BP-dependent increase in the glycoprotein's affinity to various bone matrices in a manner that exceeds the affinity produced by the previously established method, which used SMCC.
PURPOSE: To develop a novel means of conjugating bisphosphonates onto the carbohydrate moieties of glycoproteins to enhance protein affinity to bone. METHODS:1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the carbohydrate moietites of oxidized fetuin by using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (MMCCH). Bone affinity of the resulting conjugates was compared to proteins obtained from another means of conjugation, whereby aminoBP was conjugated onto fetuin's lysine moieties by using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). RESULTS: The use of the MMCCH resulted in the conjugation of up to seven aminoBPs per molecule of fetuin. These conjugates gave a 2.6-, 2.0-, 30.5-. and 1.84-fold increased affinity for untreated, ashed, demineralized bone and hydroxyapatite, respectively, as compared to conjugates from the SMCC reaction. Both conjugates exhibited a pH-independent, equally slow degradation in adult bovine serum-containing media. CONCLUSION: The use of the MMCCH chemistry to conjugate aminoBP onto fetuin was feasible. Furthermore, the described processes of conjugation resulted in amino-BP-dependent increase in the glycoprotein's affinity to various bone matrices in a manner that exceeds the affinity produced by the previously established method, which used SMCC.
Authors: Melanie Szweras; Danmei Liu; Emily A Partridge; Judy Pawling; Balram Sukhu; Cameron Clokie; Willi Jahnen-Dechent; Howard C Tenenbaum; Carol J Swallow; Marc D Grynpas; James W Dennis Journal: J Biol Chem Date: 2002-03-18 Impact factor: 5.157