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Title: The characterisation of mammalian PHOSPHO1 : an enzyme involved in bone mineralisation?
Author: Roberts, Scott John
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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Skeletal mineralisation is dependent on the generation of inorganic phosphate (Pi), which has traditionally been attributed to tissue non-specific alkaline phosphatase (TNAP). However, evidence exists to suggest the presence of other Pi generating phosphatases in bone. The most compelling being that initial bone mineralisation events in newborn TNAP knockout mice appear to be normal, although abnormalities of the skeleton and dentition appear later. PHOSPHO1 is a phosphatase, which belongs to the haloacid dehalogenase (HAD) superfamily of magnesium-dependent hydrolyases. The work of this thesis has shown that PHOSPHO1 is able to catalyse the hydrolysis of phosphoethanolamine (PEA) and phosphocholine (PCho), which displays favourable kinetics under optimal conditions indicating that these reactions would occur in vivo. Site directed mutagenesis of active site residues, along with molecular modelling, confirm this enzyme as a member of the HAD superfamily as well as implicating residues in substrate specific interactions. PHOSPHO1 protein is localised to the mineralising sites of the skeleton and cells of bone and cartilage in the mouse model. Further to this PHOSPHO1 is present in an active state within matrix vesicles, the epicentre of mineral formation. Modulation of PHOSPHO1 activity through siRNA gene knockdown studies and specific PHOSPHO1 inhibitors leads to a decrease in the mineralization potential of cells and matrix vesicles respectively. These data further support the hypothesis that PHOSPHO1 plays a central role in matrix mineralization, and indicates that the function of PHOSPHO1 is to sequester Pi from PEA and PCho contained within the glycerolipid membrane of matrix vesicles.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available