May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
In vitro Degradation of Human Aquaporin 0 by M–Calpain
Author Affiliations & Notes
  • H. Ma
    Integrative Biosciences, Oregon Health Science Univ SD, Portland, OR
  • L.L. David
    Integrative Biosciences, Oregon Health Science Univ SD, Portland, OR
  • M.A. Reviere
    Integrative Biosciences, Oregon Health Science Univ SD, Portland, OR
  • M. Azuma
    Senju Pharmaceutical Co. Ltd, Kobe, Japan
  • T.R. Shearer
    Integrative Biosciences, Oregon Health Science Univ SD, Portland, OR
  • Footnotes
    Commercial Relationships  H. Ma, None; L.L. David, None; M.A. Reviere, None; M. Azuma, Senju Pharmaceutical Co. Ltd E; T.R. Shearer, Senju Pharmaceutical Co. Ltd. R.
  • Footnotes
    Support  NIH Grants EY05786 and EY07755
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1846. doi:
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      H. Ma, L.L. David, M.A. Reviere, M. Azuma, T.R. Shearer; In vitro Degradation of Human Aquaporin 0 by M–Calpain . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1846.

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

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Abstract: : Purpose: Aquaporin 0 (AQP0) is the most abundant lens membrane protein. Post–translational modifications within the C terminus of human AQR0 include phosphorylation, deamidation and extensive backbone truncation. These modifications are more prevalent in aged fiber cells and potentially impact the movement of water through the lens. Calpains comprise a group of Ca2+–regulated intracellular proteases, which modulate cellular function by limited, specific proteolysis. Two classes are the ubiquitous calpains (µ– and m–calpains) and tissue–specific calpains (such as Lp82 and Lp85 predominantly expressed in lens). In rodent models, both m–calpain and Lp82 are involved in cataract formation by limited proteolysis of lens crystallins. The major cleavage occurring at the C terminus of AQP0 in rat cataract was identical to the cleavage site produced by m–calpain in vitro (1). The purpose of the current study was to determine if m–calpain (EC3.4.22.17) is also responsible for the cleavage of AQP0 occurring in aging human lens. Methods: AQP0 was purified from human lenses of 1–4 days of age by homogenization, centrifugation, urea washes, NaOH extraction, and protein determination by the bicinchoninic acid assay (Pierce, Rockford, IL). Proteins present in the membrane preparation were then cleaved by addition of recombinant rat m–calpain. Breakdown products were examined by SDS–PAGE. Major m–calpain cleavage sites in AQP0 were determined by reverse–phase HPLC and on–line LC/MS/MS analysis of peptides released from the membrane fraction. Results: The major breakdown products of human AQR0 produced by m–calpain observed by SDS–PAGE were 22–kDa and 20–kDa forms of AQP0. Mass spectrometric analysis of the major peptides released by m–calpain incubation indicated that cleavages producing the 22 and 20 kDa forms of AQP0 were between residues 252 and 253, and between residues 238 and 239 on the 263–residue AQP0 protein. Mass spectrometric analysis also revealed other cleavage sites produced by m–calpain at residues 237, 259 and 260. Conclusions: These are the first data examining the cleavage of human AQP0 by m–calpain. The cleavage site of AQP0 by m–calpain in vitro at residues 259, although minor in abundance, was identical to a truncation site occurring in aged human lens (2). The results also indicated that, in contrast to rat lens, unknown protease(s) or non–enzymatic hydrolysis produce other in vivo cleavages on AQR0 in human lens. 1. Kevin L Schey, et al (1999), Invest Ophthalmol Vis Sci. 40, 657–667 2. Lauren E Ball, et al (2004), Biochemistry 43, 9856–9865

Keywords: proteolysis • enzymes/enzyme inhibitors • calcium 

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