July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
The XFG- Associated Deaminase LOXL1 Is An Intrinsically Disordered Protein With A High Aggregative Ability.
Author Affiliations & Notes
  • J. Mario Wolosin
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Zheng Wang
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Robert Ritch
    Ophthalmology, New York Eye & Ear Infirmary of Mount Sinai, New York, New York, United States
  • Audrey M Bernstein
    Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States
  • Footnotes
    Commercial Relationships   J. Mario Wolosin, None; Zheng Wang, None; Robert Ritch, None; Audrey Bernstein, None
  • Footnotes
    Support  The MYS Family US Charitable Foundation, Mayer Family Foundation, The Research to Prevent Blindness, The Bright Focus Foundation NIH-NEI EY024942, SUNY Upstate Medical University Research Incentive Funds
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 6159. doi:
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    • Get Citation

      J. Mario Wolosin, Zheng Wang, Robert Ritch, Audrey M Bernstein; The XFG- Associated Deaminase LOXL1 Is An Intrinsically Disordered Protein With A High Aggregative Ability.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6159.

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

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Abstract

Purpose : XFG patient-derived Tenon’s fibroblasts (XFG-TFs) exhibit multiple features observed in neurodegenerative aggregopathies (PLoS1: 2016: e0157404; J Glaucoma 2018; 197-201), diseases shown to be induced by the aggregation of proteins with intrinsically disordered regions (IDRs). Since LOXL1, which is associated with exfoliation glaucoma (Science;317:1397) is found in XFG-TFs in the aggregate-processing autophagy pathway (J Glaucoma 2018; S44-S53) our purpose was to examine the IDR presence in, and aggregation propensity of, LOXL1.

Methods : In silico estimation of IDR probability (IDRprb) in LOXL1 and LOXL2 was made using multiple, methodologically unrelated intrinsic disorder prediction programs. Telomerase-immortalized skin fibroblasts sub-lines ectopically overexpressing LOXL1 (574 amino acids (aa)), LOXL2, LOXL1-mKATE2 chimera (Lx1-Kt; N-terminus (1-323) of LOXL1 + mKATE2; 556 aa), and homologs of the later with segment deletions (Δ[x-y]) from a CMV promoter, were generated using lentiviruses. Levels of LOXL2, LOXL1, and its homologs were determined by Western blot. Cells were stained for LOXL1 or LOXL2 using anti-LOXL1 Abs and Alexa488-conjugated secondary Abs. Protein aggregates were identified with Proteostat. After autophagy activation, Alexa488, or mKate2 fluorescence was examined by laser confocal and super-resolution microscopy.

Results : Several IDRprb analysis programs identified the entire N-terminus of LOXL1 (aa 1-330) as an IDR. In the 60-110 and 135-175 aa spans the IDRprb approached 100 % (e.g., scores > 0.95 in PrDos; prdos.hgc.jp). LOXL2 does not possess IDRs. The ectopic expression increased the steady state [LOXL1] by > 10-fold relative to endogenous levels but minimally affected [LOXL2]. Clumps of large vesicles (LV; > 50 nm) and amorphous aggregates accumulated in the perinucleus in > 20 % of LOXL1 (Alexa 488 and Proteostat fluorescence) and Lx1-Kt (by mKATE fluorescence) cells. Spatial relationships between aggregates LVs suggested that the former derive from LV disintegration. Aggregates were rarely seen (< 5 %) in Δ[28-95]LOXL1 or Δ[28-95] Lx1-Kt cells but were never observed in Δ[125-183] LOXL1 or Lx1-Kt or LOXL2 cells.

Conclusions : LOXL1 is a protein with a high propensity to aggregate. The aggregation propensity, which could underpin exfoliation glaucoma, originate in N-terminus IDRs.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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