Abstract
Opacities (cataracts) in the lens of the eye are a leading cause of preventable blindness. Aquaporins function as water channels, and the C-terminus is postulated as a regulatory domain. The C-terminal domain of aquaporin 0 (AQP0) develops numerous truncation sites during lens aging. The purpose of the present experiment was to determine if the calcium-activated protease m-calpain (EC 3.4.22.17) was responsible for truncation of human AQP0. AQP0 was isolated from young human donors, incubated with recombinant m-calpain, and the cleavage sites on the released peptides were determined by on-line electrospray ionization mass spectrometry. We found that four cleavage sites on human AQP0 could be tentatively assigned to m-calpain. This is the first evidence for possible calpain activity in human lens. Because the cause(s) of 17 other cleavage sites was unknown, the data also suggested that other, as yet unknown, proteases or non-enzymatic mechanisms are more active than calpain in human lens.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Amino Acid Sequence
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Aquaporins / chemistry*
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Aquaporins / classification
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Aquaporins / isolation & purification
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Aquaporins / metabolism*
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Calcium / metabolism
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Calcium / pharmacology
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Calpain / genetics
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Calpain / isolation & purification
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Calpain / metabolism
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Calpain / pharmacology*
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Chelating Agents / pharmacology
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Chromatography, High Pressure Liquid
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Egtazic Acid / pharmacology
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Electrophoresis, Polyacrylamide Gel
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Enzyme Activation / drug effects
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Eye Proteins / chemistry*
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Eye Proteins / isolation & purification
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Eye Proteins / metabolism*
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Humans
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Hydrolysis
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Infant, Newborn
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Lens, Crystalline / chemistry
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Lens, Crystalline / metabolism
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Membrane Glycoproteins / chemistry*
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Membrane Glycoproteins / isolation & purification
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Membrane Glycoproteins / metabolism*
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Molecular Sequence Data
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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Protein Processing, Post-Translational
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Protein Structure, Tertiary
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Recombinant Proteins / metabolism
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Recombinant Proteins / pharmacology
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Sequence Analysis, Protein
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Spectrometry, Mass, Electrospray Ionization
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Time Factors
Substances
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Aquaporins
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Chelating Agents
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Eye Proteins
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Membrane Glycoproteins
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Protein Isoforms
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Recombinant Proteins
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aquaporin 0
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Egtazic Acid
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Calpain
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m-calpain
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Calcium