December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Biochemical Analysis Of Native And Recombinant Myocilin In Human Trabecular Meshwork Cells And Supernatants
Author Affiliations & Notes
  • EA Hoffman
    Ophthalmology University of Arizona Tucson AZ
  • EP Ryan
    Ophthalmology University of Arizona Tucson AZ
  • BS McKay
    Ophthalmology Duke University Durham NC
  • WD Stamer
    Ophthalmology University of Arizona Tucson AZ
  • Footnotes
    Commercial Relationships   E.A. Hoffman, None; E.P. Ryan, None; B.S. McKay, None; W.D. Stamer, None. Grant Identification: NIH E412797(DS)AHAFRPB
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1055. doi:
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      EA Hoffman, EP Ryan, BS McKay, WD Stamer; Biochemical Analysis Of Native And Recombinant Myocilin In Human Trabecular Meshwork Cells And Supernatants . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1055.

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

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Abstract: : Purpose: The focus of the present study was to examine in a controlled fashion the cellular localization of native and recombinant myocilin (MYOC) in human trabecular meshwork (HTM) cells. Methods: HTM and COS-7 cells were either mock transfected or transfected with cDNAs encoding green fluorescent protein (GFP), MYOC/GFP, signal peptide (SP)/GFP, or SP/GFP/KDEL. Forty-eight hours post-transfection supernatants were collected, centrifuged at 2000g, concentrated ten-fold then centrifuged again at 14,000g. Pellets, whole cells and supernatants were solubilized separately in buffer containing 2% SDS and analyzed by SDS-PAGE. Fractionated proteins were transferred to nitrocellulose and analyzed by western blot using affinity purified anti-MYOC, anti-GFP and anti-beta-actin antibodies. Results: Native MYOC, recombinant MYOC and protein products of all control constructs were present in varied amounts in all four preparations tested (pellet 1, pellet 2, whole cell lysate and supernatant). For example, SP/GFP was found predominantly in cell supernatants, whereas GFP and SP/GFP/KDEL remained mostly in cell lysates. Interestingly, while MYOC/GFP was more prevalent in HTM cell supernatants, MYOC/GFP was detected predominantly in COS-7 cell lysates. Conclusion: The processes of transfection and heterologous expression contribute to the appearance of native and recombinant MYOC in cell supernatants. HTM cells appear to process recombinant MYOC differently than COS-7 cells.

Keywords: 601 trabecular meshwork • 528 proteins encoded by disease genes • 476 molecular biology 

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