May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
Functional Characterisation of Nyctalopin
Author Affiliations & Notes
  • S. Poopalasundaram
    Molecular Genetics,
    Institute of Ophthalmology, UCL, London, United Kingdom
  • L. Erskine
    Molecular Genetics and Visual Science,
    Institute of Ophthalmology, UCL, London, United Kingdom
  • M.E. Cheetham
    Institute of Ophthalmology, UCL, London, United Kingdom
  • A.J. Hardcastle
    Molecular Genetics,
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Footnotes
    Commercial Relationships  S. Poopalasundaram, None; L. Erskine, None; M.E. Cheetham, None; A.J. Hardcastle, None.
  • Footnotes
    Support  The Wellcome Trust
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1649. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      S. Poopalasundaram, L. Erskine, M.E. Cheetham, A.J. Hardcastle; Functional Characterisation of Nyctalopin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1649.

      Download citation file:

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

  • Supplements

Abstract: : Purpose: Nyctalopin, the protein encoded by the NYX gene which causes complete X–linked congenital stationary night blindness (NYX–CSNB), is a small leucine rich repeat proteoglycan. Mechanism of disease, and the function of nyctalopin are unknown, however electrodiagnostics of NYX–CSNB patients and the nob mouse suggest a specific ON–pathway deficit. The aim of this study was to test the hypothesis that nyctalopin is an extracellular protein that is modified by N–glycosylation and a GPI anchor. Methods: A series of expression constructs for human and mouse nyctalopin were created and used to express recombinant nyctalopin in bacteria and transiently transfect CHO cells to study nyctalopin localisation and post–translational modifications. To test for a functional GPI–anchor, transfected cells were treated with PI–PLC. Conserved amino acid sequence across species suggests that nyctalopin may be glycosylated, therefore transfected cells were also treated with PNGase F to test for post–translational N–glycosylation. Results: Immunoblotting of recombinant nyctalopin purified from bacteria, which lack the necessary mechanisms for post–translational modifications, show that His–tagged human nyctalopin lacking the signal sequence has an apparent molecular mass of approx. 50 kDa, which corresponds to the predicted molecular mass of this construct. In contrast, nyctalopin expressed in CHO cells by transient transfection migrates at approx. 70 kDa on immunoblots of cell lysates. CHO cell lysates were treated with PNGase F and an increase in mobility was observed (from 70 to 55 kDa). Human and mouse nyctalopin was detected as a punctuate staining pattern on the surface of unfixed CHO cells. When cells were fixed and permeabilised, we also detected nyctalopin staining in the ER. Treatment with PI–PLC released human nyctalopin from the cell surface but did not release mouse nyctalopin. Conclusions: The data show that human and mouse nyctalopin are post–translationally modified by N–glycosylation. Both human and mouse nyctalopin are expressed on the cell surface. Our data show that human nyctalopin has a functional GPI–anchor, however, mouse nyctalopin appears to be attached to the plasma membrane via a different mechanism.

Keywords: proteins encoded by disease genes • protein modifications-post translational 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.