Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Biochemistry

Supervisor

Dr. Harvey Goldberg

2nd Supervisor

Dr. Graeme Hunter

Joint Supervisor

Abstract

The mechanism of biomineralization is unknown. In bone, it has been proposed that an acidic phosphoprotein of the extracellular matrix (ECM) is important in the nucleation of hydroxyapatite (HA). The mineralized tissue protein, bone sialoprotein (BSP), has been shown to be a potent nucleator of HA and this activity is increased upon binding to type I collagen. The collagen-binding domain of BSP has been determined to be a highly conserved region (spanning residues 18-45); however, the area of collagen involved in this interaction is unknown.

In this study, a chemical cross-linking method was initially utilized to map the BSP-binding region on collagen. These experiments involved the incubation and interaction of five different single-cysteine recombinant BSP (1-100) mutants and type I collagen in the presence of two sulfhydryl-reactive, photoactivatable cross-linking reagents: ρ-azidophenacyl bromide (APB) and N-[4-(ρ-azidosalicylamido) butyl]-3’-(2’-pyridyldithio) propionamide (APDP). However, due to the low yield observed from SDS-PAGE in all instances, the complexes were not further characterized.

As an alternate approach, rotary-shadowing transmission electron microscopy (TEM) was used. This involved incubation of recombinant BSP (rBSP) with type I collagen, rotary-shadowing with platinum and analysis by TEM. Rotary-shadowing of BSP revealed a 10-nm globular structure that is linked to a thread-like structure of ~25 nm while type I collagen appeared as semi-flexible, rod-like structures. Combining BSP and collagen showed a locus of interaction at a site that averages approximately 31% from the N-terminus, based on the site of interaction of an antibody to the N-terminus of the collagen molecule. Due to the observed variability, BSP interacts with collagen between residues 255-375. Based on the locus of interaction, the arrangement of the collagen fibril, the highly flexible structure of BSP and the hydrophobic nature of the BSP-collagen binding interaction, we postulate that the N-terminal hydrophobic sequence (residues 18-45) of BSP binds collagen in an overlap region at a site that is rich in hydrophobic residues, while the two glutamic acid-rich regions extend into the gap region of the adjacent collagen molecule.

Within the collagen-binding domain of BSP, there is a highly conserved thrombin-cleavage site at Arg27-Tyr28. Thrombin cleaves rat BSP in the middle of the collagen-binding domain (residues 18-45), which effectively abolishes collagen-binding, implying physiological significance. To address the relevance, incubation of thrombin with BSP and fibrillar collagen and with demineralized bone chips was performed to determine if BSP was released from its complexes. Analysis with SDS-PAGE and western blotting demonstrated that rBSP bound to type I collagen fibrils is susceptible to thrombin cleavage. Similarly, endogenous BSP present in demineralized bone chips is also released from these chips with thrombin incubation. Since BSP appears to enhance osteoprogenitor cell migration and differentiation, this suggests that, upon injury, the release of BSP from bone may provide signals to initiate the repair process. In summary, the results from these studies are important in better understanding the BSP-collagen interaction and its role in bone formation and mineralization.


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