March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Post-translational Modifications of BFSP1
Author Affiliations & Notes
  • Roy A. Quinlan
    School of Biological/Biomedical Sciences, Biophysical Sciences Inst, Durham Univ, Durham, United Kingdom
  • Antal Tapodi
    School of Biological/Biomedical Sciences, Biophysical Sciences Inst, Durham Univ, Durham, United Kingdom
  • Edward W. Tate
    Department of Chemistry, Imperial College, London, United Kingdom
  • William P. Heal
    Department of Chemistry, Imperial College, London, United Kingdom
  • Alan R. Prescott
    School of Life Sciences, CHIPs and Division of Cell Biology and Immunology, Dundee University, Dundee, United Kingdom
  • Footnotes
    Commercial Relationships  Roy A. Quinlan, None; Antal Tapodi, None; Edward W. Tate, None; William P. Heal, None; Alan R. Prescott, None
  • Footnotes
    Support  Fight for Sight UK
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5595. doi:
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      Roy A. Quinlan, Antal Tapodi, Edward W. Tate, William P. Heal, Alan R. Prescott; Post-translational Modifications of BFSP1. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5595.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To determine the post-translational modifications of Bfsp1 that accompany lens fibre cell differentiation

Methods: : Lens membranes were prepared from both the cortical and nuclear regions and extracted with buffers to enrich for the cytoskeletal and integral membrane proteins. These fractions were used to identify and track differentiation related post-translational modifications in Bfsp1 and also to purify native protein and fragments. Recombinant BFSP1 and various fragments were expressed and purified from E. coli using the host strain BL21pLysS. Purification was effected by affinity chromatography using a nickel-chelate resin. These recombinantly expressed proteins were used to explore the sensitivity profile of Bfsp1 to caspases. Potential myristoylation of BFSP1 was determined using a coupled bacterial expression system, where the host BL21 DE3 strain coexpressed N-myristoyl transferase along with the Bfsp1 fragment of interest (Heal WP, Wickramasinghe SR, Leatherbarrow RJ, Tate EW. Org Biomol Chem.6(13):2308-15. PMID:18563263). The tagged proteins were detected using ‘click’ chemistry protocols to introduce a secondary label for downstream analysis. BFSP1 fragments and site specific mutants were also transiently expressed in the human lens cell line FHL124.

Results: : Bfsp1 is extensively post-translationally modified during lens fibre cell differentiation as seen from immunodetection data using site-specific antibodies. We have shown that D433 in the DVPD sequence is a substrate for both effector and initiator caspases including caspase 3. Additional cleavage sites for other caspases were identified in the tail domain of BFSP1. Proteolytic processing of BFSP1 was also detected in the human lens cell line, FHL124, which could be increased when the cells were exposed to an apoptogen. Introducing a D433A mutation into the potential caspase site reduced BFSP1 processing. Following caspase cleavage at D433, the exposed N-terminal glycine becomes a substrate for N-myristoyl transferase.

Conclusions: : BFSP1 is a substrate for both caspases and N-myristoyl transferase. It is the combined action of both that first cleaves BFSP1 and then exposes a suitable myristoylation site. The cleavage occurs at D433 in BFSP1 and G434 is then myristoylated. These data confirm the initial report of Bfsp1 myristoylation (Wang Z, Obidike JE, Schey KL. IOVS. 51(3):1565-74. PMID: 19875662), but now detail a potential mechanism for its exposure by caspase cleavage which we believe is linked to lens fibre cell differentiation. This site and its flanking sequences are conserved amongst Bfsp1 proteins from different species suggesting these post-translational modifications are important to Bfsp1 function in the lens.

Keywords: cytoskeleton • cytoskeleton • cytoskeleton 

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