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Originally published as JHC exPRESS on September 6, 2006.
doi:10.1369/jhc.6A7046.2006
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Journal of Histochemistry and Cytochemistry
Volume 55 (1): 35-42, 2007
Copyright ©The Histochemical Society, Inc.

In Vivo Functional Analysis of Polyglutamic Acid Domains in Recombinant Bone Sialoprotein

Rima M. Wazen, Coralee E. Tye, Harvey A. Goldberg, Graeme K. Hunter, Charles E. Smith and Antonio Nanci

Laboratory for the Study of Calcified Tissues and Biomaterials, Faculty of Dentistry, Université de Montréal, Montreal, Quebec, Canada (RMW,CES,AN), and CIHR Group in Skeletal Development and Remodeling, School of Dentistry, University of Western Ontario, London, Ontario, Canada (CET,HAG,GKH)

Correspondence to: Antonio Nanci, Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montreal, QC, Canada. E-mail: antonio.nanci{at}umontreal.ca

Bone sialoprotein (BSP) is an anionic phosphoprotein expressed in mineralizing connective tissues that binds to hydroxyapatite and nucleates its formation in vitro. Two polyglutamic acid regions (poly [E]) are believed to participate in these activities. The aim of this study was to evaluate the contribution of these acidic regions to the binding of prokaryote recombinant BSP (prBSPE) within an actual in vivo environment. Full-length prBSPE and prBSPE in which the poly [E] domains were replaced by polyalanine (prBSPA) were tagged with dinitrophenol (DNP). Tagged preparations comprised intact molecules and some fragmented forms. They were infused through a surgically created hole in the bone of rat hemimandibles and detected using immunogold labeling with anti-DNP antibodies. prBSPE-DNP was consistently immunodetected along exposed mineralized bone surfaces and osteocyte canaliculi at the surgical site. Few gold particles were observed on these surfaces when prBSPA-DNP was infused. Quantitative analyses showed significant differences in labeling between prBSPE-DNP (5.04 ± 0.73 particles/µm2) and prBSPA-DNP (1.37 ± 0.35 particles/µm2). These results indicate that poly [E] domains influence binding of prBSPE to surfaces presenting a mixture of mineral and proteins bathed by tissue fluids and suggest that they may similarly mediate the interaction of native BSP in the bone environment. (J Histochem Cytochem 55:35–42, 2007)

Key Words: bone sialoprotein • polyglutamic acid • mineralized matrix • binding capacity • immunocytochemistry


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