Tissue localization and solubilities of αA-crystallin and its numerous C-terminal truncation products in pre- and postcataractous ICR/f rat lenses.
OBJECTIVE
To investigate the tissue distribution and solubilities of various αA-crystallin truncation products in the cataractous ICR/f rat model.


METHODS
Rat lenses from precataractous (21-day) and postcataractous (100-day) ICR/f rats were sectioned and applied to a matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) target plate. Mass spectrometry images were collected to obtain a macromolecular profile of the abundant lens proteins. Separately, age-matched lenses were extracted into water-soluble (WS) and water-insoluble/urea-soluble (WI-US) fractions and subjected to MALDI-TOF mass spectrometry to correlate the protein solubilities with the imaging data. Protein identities were assigned by using a top-down proteomics approach on a high-resolution mass spectrometer.


RESULTS
Ten novel αA-crystallin truncation products were identified, along with six previously known αA-crystallin truncation products. Nearly all truncations exhibited nuclear localization, with larger truncated products displaying a ringlike localization that progressed outward toward the extranuclear, cortical region. The distributions were similar in both ages with the only significant difference being the amount of tissue area encompassed by a particular species with increasing age. Almost all nuclear products fractionated into the WI-US fraction, whereas the five largest extranuclear species exhibited mixed solubility.


CONCLUSIONS
A successful methodology for the sectioning and imaging of pre- and postcataractous ICR/f rat lenses has been established. Data collected from these analyses indicate that there are multiple αA-crystallin truncation products present in both pre- and postcataractous rats. Furthermore, these species have defined lenticular localizations and unique solubilities that may be a consequence of lens development and protein function within the lens environment.
Stella
David R
DR
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Floyd
Kyle A
KA
Grey
Angus C
AC
Renfrow
Matthew B
MB
Schey
Kevin L
KL
Barnes
Stephen
S
D000818:Animals; D002386:Cataract / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004195:Disease Models, Animal; D005583:Fourier Analysis; D007908:Lens, Crystalline / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D038202:alpha-Crystallin A Chain