Schulich school of Medicine and Dentistry logo Department of Biochemistry Schulich School of Medicine & Dentistry

Dr. Harvey A. Goldberg

Professor, CIHR Group in Skeletal Development and Remodelling
B.Sc. McGill University
Master of Philosophy, London
Ph.D. Alberta
Phone: 519.661.2182
FAX: 519.850.2459
E-mail: hagoldbe@uwo.ca


Structure and Function of Bone Proteins

Mineralized tissues are composite materials formed by the deposition of inorganic crystals within a preformed organic matrix.  In bone, and in similar tissues such as dentin, the mineral crystals are carbonated hydroxyapatite and the organic matrix consists principally of type I collagen with lesser amounts of non-collagenous proteins.   The matrix-mineral relationship in bone is characterized by the presence of HA crystals in the hole zones of the collagen fibrils and by their orientation parallel to the fibril axis.  It has been proposed that the nucleation of hydroxyapatite in bone and dentin is mediated by an anionic protein likely a phosphoprotein bound to type I collagen fibrils.  Studies conducted in our lab have demonstrated that bone sialoprotein (BSP) is likely to be this protein in bone.  

BSP is a phosphorylated sialoprotein of ~300 amino acid residues, containing sulfated tyrosine residues, an RGD (Arg-Gly-Asp) cell attachment sequence, high contents of acidic amino acids (Asp and Glu) and 2  N-linked and ~20 O-linked oligosaccharides.  Mammalian BSP also contains two highly conserved contiguous sequences of glutamic acid residues located in two glutamic acid rich domains in the amino-terminal half of the molecule.  BSP is highly specific to mineralized connective tissues.  It is also found in hypertrophic cartilage, where its expression correlates with the onset of mineralization.  In addition several types of carcinomas that metastasize to bone (e.g. breast and prostate tumours) express BSP with levels of expression that correlate with metastatic potential.  In mineralized tissues, BSP expression is localized to areas of de novo mineral formation.  By in situ hybridization BSP is found in osteoblasts actively engaged in bone formation and is not expressed in other regions of mineralized tissues.  Based on this information it is likely involved with early mineral formation in vivo.  Recent data has also shown that BSP has other important regulatory roles in bone cell biology, including cell attachment, migration and signaling.  The delineation of the effects of BSP and other mineral associated proteins including osteopontin, on skeletal cells has become a major focus of the lab.  

The studies conducted in my lab on the role of BSP and other related mineralized tissue proteins are done for the most part in collaboration with members of the CIHR Group in Skeletal Development and Remodeling.  In collaboration with Dr. Graeme Hunter, we have shown that BSP nucleates hydroxyapatite formation in vitro and that this activity is mediated by the glutamic acid-rich domains in BSP.  We have also determined the collagen-binding site in BSP and the mechanism of interaction with collagen.  Protein expression systems, for both wild type and mutated proteins, in prokaryotes and eukaryotic cells are used extensively to study the functional properties as discussed below.  

Current Research in the Lab  

Determination of Hydroxyapatite Nucleating Domains of BSP

A series of BSP mutants with alterations of the contiguous glutamic acid sequences have demonstrated that charge and possibly conformation contribute to nucleation activity.  Further mutagenesis of adjacent regions may provide additional insight into the structure-function relationship of nucleating potential.   

Collagen Binding and Mineral Formation   

Several studies have shown that BSP interacts with collagen.  We have determined the collagen binding site and the general mechanism of interaction.  Current efforts are towards determining the site on collagen that interacts with BSP, and determining the nucleation potency of BSP in the presence of collagen.  Based on these ongoing studies, we have initiated novel in vivo studies on bone regeneration.  

Cell Binding and Signaling

Current projects are on the role of BSP and other related proteins on cell attachment and signaling in a variety of skeletal cells.  With Drs. Hunter, Frank Beier and T. Michael Underhill (members of the CIHR Group) ongoing studies on osteoprogenitor and mature osteoblasts have shown a direct role of BSP in enhancement of differentiation, migration and activation of specific signaling pathways.  Similar studies are currently being conducted on chondrocytes with Dr. Beier.  In addition, we have collaborative studies with Drs. S. Jeff Dixon and Stephen Sims on determining the effects of these matrix proteins on osteoclast function (cells that resorb mineralized tissues), with a central hypothesis that these matrix proteins are involved in the activation of these cells.  For these and other projects, specific transgenic and conditional knockout mice are currently being developed.  

Structure Determination

The determination of conformation motifs in BSP is important in the elucidation of the mechanisms of nucleation, cell-matrix interactions in bone and for determining potential therapeutic reagents to promote bone and dentin formation. While the protein has been shown to be highly flexible in nature with little secondary structure, mutagenesis of the nucleating domains that alter putative structure have profound effects on nucleating potential.  Future studies will involve structural analysis of specific domains and protein-mineral modeling to relate putative structure with activity.

Role of Post-Translational Modifications in BSP

A eukaryotic expression system for recombinant BSP in bone cells has been developed in order to study the role of the post-translational modifications of BSP.  Site-directed mutagenesis of the specific phosphate-containing, or oligosaccharide containing amino acid residues will allow for the determination of their functional roles of the protein.

Current Lab Personnel

**Note: All graduate students are co-supervised.

Publications