research

 

DR. HARVEY A. GOLDBERG    

B.Sc., McGill; M.Phil., London ;  Ph.D., Alberta

Professor

Department of Biochemistry

CIHR Group in Skeletal Development and Remodeling

Schulich School of Medicine & Dentistry
(519) 661-2182; FAX: (519) 850-2459; 
Email: 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
(all graduate students are co-supervised)  

Honghong Chen (Lab manager)

Zhuhong (Shirley) Shao(technician)

Jonathan Gordon (Ph.D. student; with Dr. Hunter)

Kamal Gill (Ph.D. student; with Dr. Beier)

Gurprett Singh (Ph.D. student; with Dr. Hunter)

Mahmoud Esmail-nia (M.Sc. student; with Dr. Hunter)

Wailan Chan (M.Sc. student in BME Program; with Drs. Rizkalla and Hunter)

Janel Yu (Dentistry summer research student)

Kyle Carter (summer research student)

 

Selected Recent References  

  1. Forsprecher J, Wang Z, Goldberg HA, Kaartinen MT. Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein. Cell Adh Migr. 2011 Jan-Feb;5(1):65-72. Epub 2011 Jan 1. PubMed PMID: 20864802; PubMed Central PMCID: PMC3038101.
  2. McDonald EE, Goldberg HA, Tabbara N, Mendes FM, Siqueira WL. Histatin 1 resists proteolytic degradation when adsorbed to hydroxyapatite. J Dent Res. 2011 Feb;90(2):268-72. Epub 2010 Nov 12. PubMed PMID: 21076122.
  3. Grohe B, Chan BP, Sørensen ES, Lajoie G, Goldberg HA, Hunter GK. Cooperation of phosphates and carboxylates controls calcium oxalate crystallization in ultrafiltered urine. Urol Res. 2011 Jan 14. [Epub ahead of print] PubMed PMID: 21234554.
  4. Chan WD, Goldberg HA, Hunter GK, Dixon SJ, Rizkalla AS. Modification of polymer networks with bone sialoprotein promotes cell attachment and spreading. J Biomed Mater Res A. 2010 Sep 1;94(3):945-52. PubMed PMID: 20730931.
  5. Tenboll A, Darvish B, Hou W, Duwez AS, Dixon SJ, Goldberg HA, Grohe B, Mittler S. Controlled deposition of highly oriented type I collagen mimicking in vivo collagen structures. Langmuir. 2010 Jul 20;26(14):12165-72. PubMed PMID: 20560559.
  6. Wine E, Shen-Tu G, Gareau MG, Goldberg HA, Licht C, Ngan BY, Sorensen ES, Greenaway J, Sodek J, Zohar R, Sherman PM. Osteopontin mediates Citrobacter rodentium-induced colonic epithelial cell hyperplasia and attaching-effacing lesions. Am J Pathol. 2010 Sep;177(3):1320-32. Epub 2010 Jul 22. PubMed PMID: 20651246; PubMed Central PMCID: PMC2928965.
  7. Hunter GK, O'Young J, Grohe B, Karttunen M, Goldberg HA. The Flexible Polyelectrolyte Hypothesis of Protein-Biomineral Interaction. Langmuir. 2010 Jun 8. [Epub ahead of print] PubMed PMID: 20527831.
  8. Baht GS, O'Young J, Borovina A, Chen H, Tye CE, Karttunen M, Lajoie GA, Hunter GK, Goldberg HA. Phosphorylation of Ser136 is critical for potent bone sialoprotein-mediated nucleation of hydroxyapatite crystals. Biochem J. 2010 May 27;428(3):385-95. PubMed PMID: 20377527.
  9. Azzopardi PV, O'Young J, Lajoie G, Karttunen M, Goldberg HA, Hunter GK. Roles of electrostatics and conformation in protein-crystal interactions. PLoS One. 2010 Feb 19;5(2):e9330. PubMed PMID: 20174473; PubMed Central PMCID: PMC2824833.
  10. Grohe B, Taller A, Vincent PL, Tieu LD, Rogers KA, Heiss A, Sørensen ES, Mittler S, Goldberg HA, Hunter GK. Crystallization of calcium oxalates is controlled by molecular hydrophilicity and specific polyanion-crystal interactions. Langmuir. 2009 Oct 6;25(19):11635-46. PubMed PMID: 19725562.
  11. Gordon JA, Sodek J, Hunter GK, Goldberg HA. Bone sialoprotein stimulates focal adhesion-related signaling pathways: role in migration and survival of breast and prostate cancer cells. J Cell Biochem. 2009 Aug 15;107(6):1118-28. PubMed PMID: 19492334.
  12. Gordon JA, Hunter GK, Goldberg HA. Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation. Cells Tissues Organs. 2009;189(1-4):138-43. Epub 2008 Aug 26. PubMed PMID: 18728350.
  13. Baht GS, Hunter GK, Goldberg HA. Bone sialoprotein-collagen interaction promotes hydroxyapatite nucleation. Matrix Biol. 2008 Sep;27(7):600-8. Epub 2008 Jun 24. PubMed PMID: 18620053.
  14. Gill KS, Beier F, Goldberg HA. Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein. Am J Physiol Cell Physiol. 2008 Jul;295(1):C38-49. Epub 2008 May 7. PubMed PMID: 18463228; PubMed Central PMCID: PMC2493551.
  15. Grohe B, O'Young J, Ionescu DA, Lajoie G, Rogers KA, Karttunen M, Goldberg HA, Hunter GK. Control of calcium oxalate crystal growth by face-specific adsorption of an osteopontin phosphopeptide. J Am Chem Soc. 2007 Dec 5;129(48):14946-51. Epub 2007 Nov 10. PubMed PMID: 17994739.
  16. Gordon JA, Tye CE, Sampaio AV, Underhill TM, Hunter GK, Goldberg HA. Bone sialoprotein expression enhances osteoblast differentiation and matrix mineralization in vitro. Bone. 2007 Sep;41(3):462-73. Epub 2007 May 6. PubMed PMID: 17572166.