Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Designing a magic bullet oncotoxin: manipulating the lewis antigen specificity of lectinolysin to target lewis y-associated tumours (#145)

Bronte A Johnstone 1 2 , Sara L Lawrence 2 , Jessica K Holien 2 , Craig J Morton 1 2 , Rodney K Tweten 3 , Michael W Parker 1 2
  1. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria, Australia
  2. ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
  3. Department of Microbiology and Immunology, University of Oklahoma, Oklahoma City, OK 73104, USA

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins produced by more than 50 species of Gram positive bacteria. Secreted as soluble monomers, CDCs oligomerise into large circular prepores on the surface of target cell membranes, a process dependent on recognition of membrane cholesterol. Various structural changes and transitions results in insertion of β-hairpins into the lipid bilayer, forming a large β-barrel pore greater than 250 Å in diameter that results in cell lysis. Lectinolysin (LLY) is an atypical CDC, possessing an additional amino terminal domain, referred to as D0 or the lectin domain (LLYlec). Previous research elucidated a distinctive recognition for the difucosylated Lewis b (Leb) and Lewis y (Ley) antigens (Farrand et. al., Biochemistry (2008) 47, 7097-7107). Ley antigen expression is upregulated in various endothelial tumours, and therefore has been proposed as a promising target for cancer therapy. LLY is a potential oncotoxin with inherent specificity for Ley antigen, with therapeutic potential if Leb cross-reactivity were to be eliminated. Here, we report the characterisation of four LLYlec mutants: LLYlec Y62R, Y62F, Y62K and Y62W. Structures of LLYlec wt and LLYlec Y62F in complex with Lewis antigens are described. Preliminary binding studies, using differential scanning fluorimetry (DSF), revealed increased Ley selectivity of LLYlec Y62W, compared to wild-type. Future experiments include ongoing X-ray crystallography, and further investigation of Lewis antigen binding using Surface Plasmon Resonance (SPR). Implications of preliminary data will be further discussed, with research contributing to the development of a novel potential cancer therapeutic for the treatment of epithelial tumours with a Ley phenotype.

  1. Farrand, S., Hotze, E., Friese, P., Hollingshead, S.K., Smith, D.F., Cummings, R.D., Dale, G.L., and Tweten, R.K. (2008) Characterization of a streptococcal cholesterol-dependent cytolysin with a Lewis y and b specific lectin domain. Biochemistry. 47, 7097-7107