Posttranslational modifications of the bovine lens beaded filament proteins filensin and CP49.
OBJECTIVE
The lens beaded filament proteins filensin and CP49 are phosphorylated proteins that undergo proteolytic degradation with fiber cell age; however, the specific sites of modifications remain largely unknown. The purpose of this study was to identify posttranslational modifications (PTMs) in bovine lens beaded filament proteins.


METHODS
Filensin and CP49 were enriched by urea extraction of lens fiber cell homogenates after the water-soluble fraction was removed. The urea-soluble fraction was separated by SDS-PAGE, and the corresponding filensin and CP49 bands were digested by trypsin, Lys C, or Glu C. The enzymatic digests were analyzed by HPLC mass spectrometry.


RESULTS
The sequences of lens beaded filament proteins were systematically mapped, and putative database sequence errors of filensin were identified. The data also indicated that Met-1 of CP49 was removed and Ser2 was acetylated. Nine phosphorylation sites on filensin and seven phosphorylation sites on CP49 were identified. Filensin was found to be truncated at D431 and L39, and the resulting new N termini were N-myristoylated and N-acetylated, respectively. Truncation of CP49 occurred at D37. Aspartic acid isomerization to isoaspartic acid occurs at the major truncation sites of filensin (D431) and of CP49 (D37).


CONCLUSIONS
This study identified sites of phosphorylation and truncation in filensin and CP49 and revealed two unusual PTMs: postproteolytic N-acetylation and N-myristoylation of filensin. The detailed knowledge about these PTMs provides important information for further study of their functional consequences-for example protein redistribution during lens fiber cell differentiation and aging.
Wang
Zhen
Z
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232-8575, USA.
Obidike
Joy E
JE
Schey
Kevin L
KL
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D026581:Isoaspartic Acid / Q000737:chemistry; D007536:Isomerism; D007908:Lens, Crystalline / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry
D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D026581:Isoaspartic Acid; C079098:filensin; C084808:phakinin; D001224:Aspartic Acid