Increased sensitivity of amino-arm truncated betaA3-crystallin to UV-light-induced photoaggregation.
OBJECTIVE
Exposure to UV-B light (wavelength, 290-320 nm) is a well-documented risk factor for age-related cataracts. As the lens ages, beta-crystallins tend to undergo proteolytic cleavage of their terminal extensions. To delineate the effects of loss of terminal arms on beta-crystallin function, the sensitivity of purified recombinant wild-type (rbetaA3) to UV-irradiation induced aggregation was compared with that of betaA3-crystallin missing the N-terminal extension (rbetaA3tr).


METHODS
Proteins were expressed in baculovirus-infected Sf9 cells and purified by chromatography. Purified protein solutions (pH 7.4) were reduced by using Tris (2-carboxyethyl) phosphine HCl and irradiated with a 308-nm excimer laser at physiologically relevant UV doses and wavelengths (308 nm), and light-scattering (633 nm) was measured. Irradiated crystallins were analyzed by matrix-assisted desorption ionization (MALDI) and tandem liquid chromatography/mass spectrometry (LC-MS/MS).


RESULTS
UV-irradiation of both rbetaA3 and rbetaA3tr resulted in major loss of soluble protein, as shown by absorption at 280 nm, size-exclusion chromatography (SEC) and SDS-PAGE, with concomitant formation of insoluble aggregates producing light-scattering. Compared with wild-type rbetaA3, rbetaA3tr showed a significant tendency to begin scattering light at lower UV dose and had a higher aggregation rate with increasing UV exposure. Changes in irradiated crystallins include aggregation and cross-linking, photolysis, and oxidation of methionine and tryptophan residues.


CONCLUSIONS
Loss of beta-crystallin terminal arms appears to increase their tendency to aggregate in response to UV irradiation, suggesting that this loss in the maturing lens may increase susceptibility to age-related cataract.
Sergeev
Yuri V
YV
National Eye Institute, National Institutes of Health, Building 10, Room 10B10, 10 Center Drive, Bethesda, MD 20892, USA. sergeev@helix.nih.gov
Soustov
Lev V
LV
Chelnokov
Eugene V
EV
Bityurin
Nikita M
NM
Backlund
Peter S
PS
Wingfield
Paul T
PT
Ostrovsky
Mikhail A
MA
Hejtmancik
J Fielding
JF
D000375:Aging / Q000473:pathology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002386:Cataract / Q000473:pathology; D002850:Chromatography, Gel; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008027:Light; D008715:Methionine / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D010777:Photochemistry; D010782:Photolysis; D011489:Protein Denaturation / Q000528:radiation effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014364:Tryptophan / Q000378:metabolism; D014466:Ultraviolet Rays; D038205:beta-Crystallin A Chain
C051374:Cryba1 protein, mouse; D003459:Crystallins; D011994:Recombinant Proteins; D038205:beta-Crystallin A Chain; D014364:Tryptophan; D008715:Methionine