SFI UREKA Site in Molecular Recognition
Translating Design into Application

Biological Studies

Project 13:      Investigating Key Aspects of the Interaction Between a C-type lectin Expressed by Bone Cells with the b -glucan, Laminaran

Supervisor:     Dr. Maria Tuohy, Department of Biochemistry

Research Project: Polysaccharides from marine plants, such as algae, have been identified as an interesting group of biological response modifiers (BRMs) ¹. Some of the suggested bioactivities of these BRMs range from anti-microbial to anti-cancer effects.  Bioactivity depends on the composition, size and charge properties of these carbohydrates (CHOs), properties that can be altered by changes in seasonal and environmental conditions, as well as by extraction methods ^2-4.  Our studies have focused on developing novel extraction methods to maximize native structure, on gaining a greater understanding of the effects of selected CHOs from brown algae on mammalian cells, the characterisation of potential bioactive properties, using enzymes to enhance bioactivity and on strategies to decipher the mechanism of action of the target biomolecules ⁴.   Some of this work has recently led to the discovery of novel therapeutic possibilities for the b-1,3(6)glucan, laminaran ^5,6 as treatment with this laminaran up-regulates human b-glucan receptor (hbGR; the human homologue of dectin-1) expression in SaOS-2 osteosarcoma cells and human mesenchymal stem cells ⁴.

The student researcher will generate and isolate a range of oligosaccharides from laminaran by combining selective hydrolysis with purified b-1,3-glucanase from the fungal species T. emersonii with downstream separation of the oligosaccharides by HPLC. He/She will grow SaOS-2 cells in culture, treat the cells with a concentration of the MGBG laminaran and measure effects on cell morphology, survival and proliferation and on the expression of key housekeeping genes and biomarkers. The student will also re-confirm expression of the hbGR by RT-PCR and Western blotting.  Binding of the hbGR to its ligand will be confirmed using confocal and fluorescence microscopy and flow cytometry with labeled laminaran (e.g. FITC-laminaran).  The ability of individual oligosaccharide fractions (as well as unlabelled laminaran) to compete with binding of labeled laminaran to the hbGR will be investigated to gain information on the unit-ligand for bioactivity.

The student will work closely with senior PG and Post-doctoral researchers on a daily basis and will be trained in a range of laboratory skills. The student will gain experience in glycobiology, glycoengineering, as well as in cell and molecular biology. Training will include cell culture, microscopy and flow-cytometry, methods to assess cell proliferation and viability, isolation of nucleic acids and proteins, PCR and RT-PCR, immunofluorescence and immunoprecipitation, nucleic acid and protein electrophoresis, using enzymes to generate oligosaccharides, HPLC

References: [1] Leung, et al., (2006) Immunol. Lett. 105: 101-114. [2] Hennequart, et al., (2004) Aqua Feeds: Formulation & Beyond, 1: 14-18. [3] Zvyagintseva et al., (2003) J. Exp. Mar. Biol. Ecol. 294: 1-13. [4] Kelly, S. (2006) Ph.D. Thesis. National University of Ireland, Galway [5] Ryan, C. (2007) Ph.D. Thesis. NUI, Galway. [6] O’Sullivan et al., (2003) BioNet Conference, GMIT, Galway, Ireland.

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