Proteomic and sequence analysis of chicken lens crystallins reveals alternate splicing and translational forms of beta B2 and beta A2 crystallins.
OBJECTIVE
To characterize the adult chicken lens proteome using mass spectrometry and two-dimensional gel electrophoresis (2-DE).


METHODS
Lens proteins from 10-week old chickens were separated by gel filtration and reversed-phase chromatography, and whole protein masses were measured with electrospray mass spectrometry. Water-soluble lens proteins were separated by 2-DE and identified by tandem mass spectrometry of in-gel digests.


RESULTS
Whole protein masses were consistent with all major chicken lens crystallin sequences, except for beta B2 and beta B3. Subsequent cDNA sequencing revealed errors in published sequences translating into 2- and 7-amino-acid differences, respectively, for beta B2 and beta B3, which were in better agreement with the measured masses. Previously uncharacterized forms of beta A2 and beta B2 were observed. The novel form of beta A2 had four fewer amino acids, was more abundant, and resulted from translation at a second start codon. The novel form of beta B2 contained 14 additional amino acids in the interdomain linker and resulted from alternate splicing within intron 4 of the transcript. All examined crystallins, except beta A3, for which data could not be obtained, were N-terminally acetylated, and all beta-crystallins lacked an initial methionine, except for the smaller beta A2 form. In-gel digests identified 29 proteins on the 2-DE map and indicated that truncation occurs within N-terminal extensions of beta-crystallins during lens maturation.


CONCLUSIONS
The complementary techniques 2-DE, mass spectrometry, and DNA sequencing were used to provide the most complete description of the adult chicken lens proteome to date and identified alternate forms of beta A2 and beta B2.
Department of Integrative Biosciences, Oregon Health and Science University, Portland, 97239, USA.
Phillip A
PA
Wilmarth
Jennifer R
JR
Taube
Michael A
MA
Riviere
Melinda K
MK
Duncan
Larry L
LL
David
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000235:genetics*; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D007908:Lens, Crystalline / Q000737:chemistry*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D038205:beta-Crystallin A Chain / Q000737:chemistry / Q000235:genetics*; D038206:beta-Crystallin B Chain / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics*
D038205:beta-Crystallin A Chain; D038206:beta-Crystallin B Chain; C113634:beta-crystallin B2