Associate Professor Clem Robinson

Telephone: +61 - 3 9905 3775 Facsimile: +61 - 3 9905 4699 Email: Clem.Robinson@med.monash.edu.au

Current Research Interests:

Research Links:

Sulphite formation and calgranulins

Biosynthesis and Turnover of Cartilage Aggrecan.

The mechanism of synthesis of cartilage proteoglycan including the use of beta-xylosides and beta-D-galactosides as chain initiators of glycosaminoglycan synthesis. The regulation of galactosyltransferase I and galactosyltransferase II - enzymes which respectively catalyse the transfer of the second and third monosaccharide residues to the growing polysaccharide chain. The control of proteoglycan core protein synthesis and polysaccharide chain initiation. The enzymic mechanism of aggrecan breakdown in articular cartilage in ageing and arthritis.

Sulphite metabolism in mammalian polymorphonuclear neutrophils (PMN).

The mechanism of reduction of inorganic sulphate to inorganic sulphite by these cells. The role of calgranulin C in sulphate metabolism and the role of sulphite in the biological function of neutrophils.

Recent publications:

1. Yang, Z., deVeer, M. J., Gardiner, E. E., Devenish, R. J., Handley, C. J., Underwood J. R. & Robinson, H. C. (1996). Rabbit polymorphonuclear neutrophils form ³⁵S-labeled S-sulfo-calgranulin-C when incubated with inorganic [³⁵S]sulfate. J. Biol. Chem. 271, 19802 - 19809.
   
   
2. Ilic, M. Z., Robinson, H. C. and Handley, C. J. (1998). Characterization of Aggrecan Retained and Lost from the Extracellular Matrix of Articular Cartilage, J. Biol. Chem. 273, 17,451 - 17,458.
   
   
3. Tester, A. M., Ilic, M. Z., Robinson, H. C. and Handley, C. J. (1999). Metabolic processing of newly synthesised link protein in bovine articular cartilage explant cultures, Matrix Biology, 18, 65 - 74.
   
   
4. Winter, A. D., Campbell, M. A., Robinson, H. C. and Handley, C. J. (2000). Catabolism of newly synthesised decorin by explant cultures of bovine ligament, Matrix Biology, 19, 129-138.
   
   
5. Ilic, M. Z. Vankemmelbeke, M. N. Holen, I. Buttle, D. J., Robinson, H. C. and Handley, C. J. (2000). Bovine joint capsule and fibroblasts derived from joint capsule express aggrecanase activity. Matrix Biology. 19, 257-265.

Current Teaching:

Convenor of Biochemistry in human function (BMS2021) which is a core second year subject in the degree Bachelor of Biomedical Science.

Lecturing commitments in Cells, tissues and organisms (BMS1021), The molecules of life (BCH2011), Molecular biology of the cell (BCH3022), and Biochemistry and molecular biology (BCH4000).

• Biochemistry in Human Function
• Bachelor of Biomedical Science
• Cells, tissues and organisms
• The molecules of life
• Molecular biology of the cell
• Biochemistry and molecular biology

Current Research Projects for Honours/postgraduate students.

1. Sulphite formation by rabbit polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMN) are phagocytic white blood cells, which migrate to sites of tissue injury and play a pivotal role in host defence against infection. Work in my laboratory has demonstrated the production and release of inorganic sulphite by both circulating and extravascular (activated) rabbit PMN and we have established that these cells also secrete S-sulphocalgranulin C, a sulphated derivative of a soluble endogenous protein of PMN and a member of the S100 protein family. The hypothesis to be tested in this project is that sulphite production and secretion is an important part of the PMN cell’s armory against bacterial infection.

The aims of this project are to investigate the molecular events associated with sulphite production by PMN and to determine the role of the S-sulphation of calgranulin C in this process.

This project will involve isolation of rabbit PMN by intraperitoneal lavage, incubation of the PMN with inorganic [³⁵S]sulphate in vitro for short periods of time to detect the macromolecular components which first become radio-labelled. These experiments will be carried out in the presence and absence of a charged maleimide (maleimidopropionic acid) to trap radioactively labelled extracellular sulphite and extracellular proteins. Labelled intracellular proteins will be trapped by the subsequent addition of uncharged maleimide (N-ethylmaleimide). Labelled proteins will be isolated by FPLC in combination with SDS-PAGE and after purification their N-terminal amino acid sequence will be determined to establish their identity. The amino terminal amino acid sequence of the proteins will be used to search for the parent protein by data base searching and comparison with known bacterial and plant PAPS reductases which are known to produce sulphite in these organisms.

Techniques to be used: use of radioactive isotopes, cell isolation techniques, protein purification by ion-exchange FPLC, gel-filtration, hydrophobic interaction chromatography, SDS-PAGE, data base searching.

This project will involve working in association with members of the research team investigating aspects of autoimmunity and participating in that group's seminar programme.

2. Assay of aggrecanase activity in synovial joint tissues.

This project will investigate the mechanism of degradation of large aggregating proteoglycans (hyalectins), namely aggrecan and versican, present in the extracellular matrix of connective tissues that make up synovial joints. Of particular interest is the involvement of the specific proteinase aggrecanase in this process. Aggrecanase, has been implicated in the catabolism of aggrecan in both normal and arthritic articular cartilage.

The aims of this project are to establish a cell-free assay for aggrecanase activity in culture medium from joint capsule cultures. Such as assay will then enable ongoing work into the mechanism by which joint capsule, ligament and tendon degrade the large aggregating proteoglycans present within them. This investigation will compare the degradation of the large aggregating proteoglycans by unfractionated conditioned medium from explant joint tissue, that is known to exhibit aggrecanase-like activity, with that brought about by purified aggrecanase, in an attempt to demonstrate that aggrecanase is the primary (and perhaps only) protease involved in aggrecanase degradation. The work will also investigate the synthesis of aggrecanase activity by explant cultures of joint capsule, ligament and tendon and cultured cells derived from these tissues, and ultimately purify aggrecanase from conditioned medium of explant cultures of joint capsule and determine its primary structure.

The Hypotheses to be tested in this project are: a) that aggrecanase plays a pivotal role in the catabolism of large aggregating proteoglycans in synovial joints, and b) that the synthesis and release of aggrecanase by capsular tissue in vivo contributes to the loss of aggrecan from articular cartilage in arthritis.

Techniques to be used: the use of radioactive isotopes and fluorescent probes, tissue extraction techniques, protein purification by ion-exchange FPLC, gel-filtration, hydrophobic interaction chromatography, SDS-PAGE, polyclonal antibody production, ELISA assays and Western blotting.

This project will involve working in association with members of research teams investigating aspects of autoimmunity and of cartilage and arthritis (Prof. Chris Handley's research group at School of Human Biosciences, La Trobe University).