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Investigating protein glycosylation in bile duct cells

Primary Supervisor: Dr Padma Sheela Jayaraman, Institute of Cancer and Genomic Sciences

Secondary supervisor: Professor Paula Mendes

PhD project title: Investigating protein glycosylation in bile duct cells

University of Registration: University of Birmingham

Project outline:

    AIM

    To understand changes in the secreted glycoproteins produced by primary bile duct cells.

    BACKGROUND

    Bile duct diseases including cholangitis and fibrosis are examples of complex diseases that involve a combination of genetic and environmental factors. Bile duct wounding, by exposure to toxins, metabolites (high bile acids/oxysterols), and /or pathogens (viruses or liver flukes), leads to a wound-healing response involving recruitment and activation of inflammatory immune cells, release of cytokines and activation of stromal cells and vasculature. Unregulated wound healing promotes cholangiocyte proliferation and stromal cell dysregulation (fibrosis) and is accompanied by changes in expression and glycosylation of mucin proteins. 

    Mucins are secreted by bile duct cells to protect the bile tract. The MANA1 gene encodes the enzyme that regulates N-glycosylation of proteins by regulating the first step in the enzymatic chain that leads to complex mannose glyosylation of proteins such as mucins. Reduction of MANA1 leads to increases of an intermediate in mannose glycosylation (mannose high M6N2) at the cell membrane and also to increased presence of mannose high mucin proteins present in serum. The transcription factor PRH represses the expression of MAN1A1 and therefore this protein can potentially regulate N-glycosylation of multiple proteins including mucins (current data). Changes in PRH and MAN1A1 will lead to alterations in bile duct cell behaviour and wound healing.

    HYPOTHESIS

    Elevated PRH activity will lead to increased mannose high N-glycosylation of mucins present at the cell surface and also in serum.

    EXPERIMENTAL METHODS AND RESEARCH PLAN

    • Isolation of primary bile duct cells and from primary human liver using magnetic antibody based technology. We have experience of purifying bile duct cells, hepatocytes and stromal Hepatic Stellate Cell fibroblasts from human liver. This resource and technology is uniquely available in the Jayaraman/Afford laboratories. Using transcriptomics approaches including RNA sequencing and Global Histone ChIP sequencing we will investigate the consequences of bile acids, and changes in expression of the PRH transcription factor on MAN1A1 and protein glycosylation on the behaviour of bile duct cells.
    • Professor Paula Mendes is expert in the detection of protein glycosylation using multiple technologies. Her group has developed a synthetic modular approach to create molecularly casted fluorescent nanoparticles with high selectivity for glycosylated proteins. The synthetic approach allows the creation of nanopockets on the nanoparticle surface with carbohydrate receptors spatially arranged in a specific manner to fit only the target glycoprotein glycoform. The technology will be used in this project to detect specifically particular high-mannose glycoprotein glycoforms. The technology overcomes the partial recognition associated with lectins and the immunogenicity issues associated with antibodies. The strategy can effectively be implemented to recognise the entire glycan target and be adapted to a broad range of glycans.

    BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing

      Techniques that will be undertaken during the project:

      • Isolation of bile duct cells from liver
      • Lentiviral transduction
      • Proliferation assays
      • Viability assays
      • RNA sequencing
      • Quantitative PCR
      • Western blotting
      • In vitro-Glycobiology
      • Nanoparticle synthesis
      • Transmission electron microscopy
      • Isothermal Titration Calorimetry

      Contact: Dr Padma Sheela Jayaraman, University of Birmingham