BIOCHEMICAL AND BIOLOGICAL CHARACTERISTICS OF NOVEL PISCINE FORMS OF PARATHYROID HORMONE AND PARATHYROID HORMONE RELATED PEPTIDE IN FISH : A PROTEOMIC APPROACH

Funder

Organizational Unit

Publications

Biochemical and biological characteristics of novel piscine forms of parathyroid hormone and parathyroid hormone related peptide: a proteomic approach

Publication . Anjos, Liliana; Power, Deborah

Vertebrate bone remodeling and development is a fundamental physiological process which is regulated by multiple factors. Homologues of mammalian parathyroid hormone (PTH)/ parathyroid hormone related peptide (PTHrP) which have a role in skeletal homeostasis have been identified in teleosts but their function is poorly understood. This dissertation describes the biochemical characterization of PTH/ PTHrP family from an advanced teleost, sea bream. Recombinant His-tag PTHA\PTHB and PTH-L proteins were produced using a prokaryotic expression system. In silico analysis predicted a secondary structure for PTHA and PTHB composed of -helix and random coils which was confirmed by biophysical studies of the recombinant proteins. A reference proteome of acellular sea bream bone was generated using 2D electrophoresis coupled to MALDI-TOFF analysis and the way in which sea bream recombinant PTHrP modifies the bone proteome was established. Approximately 300 proteins were mapped in the bone proteome and 125 proteins were analysed of which 94.4% matched proteins in public databases (118). Comparison of control and PTHrP treated fish revealed 8 differentially expressed proteins and the results suggest that global proteome analysis identified novel PTHrP regulated pathways in fish bone. To establish the physiological function of piscine CRTAC2 (Cartilage acidic protein 2), which is a homologue of mammalian CRTAC1 (chondrocyte marker) a recombinant protein was produced. The secondary structure of the soluble recombinant protein contained 9% -helix, 32% - sheet and 58% unordered protein and it had a hyperthermostable tertiary structure. Western blot revealed CRTAC had a widespread tissue distribution and was abundantly expressed in kidney and liver and that the native protein exists in tissue extracts as high molecular weight aggregates. Preliminary physiological studies in fish revealed that sbCRTAC2 in common with collagen I favors outgrowth of epithelia and highlights a possible function which will be studied in the future.

2010Doctoral thesis

Restricted access