June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Collagen binding site in SPARC is required for chaperone activity
Author Affiliations & Notes
  • Brian James Liu
    Case Western Reserve University, Cleveland, Ohio, United States
  • Caroline Christina Sachse
    Case Western Reserve University, Cleveland, Ohio, United States
  • Min Hyung Kang
    Case Western Reserve University, Cleveland, Ohio, United States
  • Douglas J Rhee
    Case Western Reserve University, Cleveland, Ohio, United States
    Ophthalmology and Visual Sciences, University Hospitals, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Brian Liu, None; Caroline Sachse, None; Min Hyung Kang, None; Douglas Rhee, None
  • Footnotes
    Support  5 RO1 EY019654
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 1010. doi:
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      Brian James Liu, Caroline Christina Sachse, Min Hyung Kang, Douglas J Rhee; Collagen binding site in SPARC is required for chaperone activity. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1010.

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

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Abstract

Purpose : Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein that regulates extracellular matrix (ECM) turnover in the trabecular meshwork (TM) and modulates intraocular pressure. Overexpression of SPARC in human TM cells leads to increased levels of ECM proteins as a result of chaperone activity in SPARC protecting ECM proteins from degradation by matrix metalloproteinases (MMPs). We hypothesize that this chaperone activity requires SPARC-collagen binding at the specific binding site.

Methods : Site-specific mutated SPARC (R149A) constructed in adenovirus was overexpressed in human TM cells and purified. In vitro MMP assays were performed in a reaction mixture with mutated SPARC, exogenous MMP-2 or MMP-9, collagen I and Ca2+ (0 or 2 mM) for 6 hours at 37 C. Immunoblot analysis was performed to quantify the undigested collagens.

Results : Mutated SPARC did not demonstrate a chaperone effect on collagen I. When incubated with MMP-2 in 2mM Ca2+ buffer, the relative amount of collagen retained was 0.46±0.15 for H2O, 0.36±0.11 for BSA, and 0.30±0.10 for SPARC (p=0.374, n=5, H2O vs. SPARC), (p=0.661, n=5, BSA vs. SPARC). When incubated with MMP-9 in 2mM Ca2+ buffer, the relative amount of collagen retained was 0.13±0.04 for H2O, 0.43±0.23 for BSA, and 0.48±0.11 for SPARC (p=0.026, n=4, H2O vs. SPARC), (p=0.835, n=4, BSA vs. SPARC). The relative amount of collagen retained when incubated with MMP-2 in 0mM Ca2+ buffer was 0.36±0.19 for H2O, 0.59±0.09 for BSA, and 0.65±0.06 for SPARC (p=0.178, n=5, H2O vs. SPARC), (p=0.578, n=5, BSA vs. SPARC). The relative amount of collagen retained when incubated with MMP-9 in 0mM Ca2+ buffer was 0.75±0.37 for H2O, 0.51±0.09 for BSA, and 1.15±0.47 for SPARC (p=0.532, n=4, H2O vs. SPARC), (p=0.228, n=4, BSA vs. SPARC).

Conclusions : Our results demonstrate that the modification of the collagen binding site in SPARC diminishes the chaperone activity of SPARC on collagen I against MMP-mediated degradation. This finding suggests that the collagen-binding site in SPARC plays a critical role for chaperone activity against MMP-mediated degradation.

This is a 2020 ARVO Annual Meeting abstract.

 

Fig. 1: Typical immunoblot results of chaperone assay containing MMP-2 and 0mM Ca2+

Fig. 1: Typical immunoblot results of chaperone assay containing MMP-2 and 0mM Ca2+

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