Dataset Information

Posttranslational modifications of the bovine lens beaded filament proteins filensin and CP49.

ABSTRACT:

Purpose

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.

SUBMITTER: Wang Z

PROVIDER: S-EPMC2868428 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Publications

Posttranslational modifications of the bovine lens beaded filament proteins filensin and CP49.

Wang Zhen Z Obidike Joy E JE Schey Kevin L KL

Investigative ophthalmology & visual science 20091029 3

<h4>Purpose</h4>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.<h4>Methods</h4>Filensin and CP49 were enriched by urea extraction of lens fiber cell homogenates after the water-soluble fraction was removed. The urea-sol ...[more]

PMID: 19875662

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Project description:BackgroundDue to the unique nature of spermatozoa, which are transcriptionally and translationally silent, the regulation of capacitation is based on the formation of posttranslational modifications of proteins (PTMs). However, the interactions between different types of PTMs during the capacitation remain unclear. Therefore, we aimed to unravel the PTM-based regulation of sperm capacitation by considering the relationship between tyrosine phosphorylation and reversible oxidative PTMs (oxPTMs), i.e., S-nitrosylation and S-glutathionylation. Since reversible oxPTMs may be closely related to peroxyredoxin (PRDX) activity, the second aim was to verify the role of PRDXs in the PTM-based regulation of capacitation.MethodsCryopreserved bull sperm were capacitated in vitro with or without PRDX inhibitor. Qualitative parameters of sperm and symptoms characteristic of capacitation were analyzed. Posttranslational protein modifications (S-nitrosylation, S-glutathionylation, tyrosine phosphorylation) were investigated at the cellular level (flow cytometry, fluorescence microscopy) and at the proteomic level (fluorescent gel-based proteomic approach).ResultsZona-pellucida binding proteins (ACRBP, SPAM1, ZAN, ZPBP1 and IZUMO4) were particularly rich in reversible oxPTMs. Moreover, numerous flagellar proteins were associated with all analyzed types of PTMs, which indicates that the direction of posttranslational modifications was integrated. Inhibition of PRDX activity during capacitation caused an increase in S-nitrosylation and S-glutathionylation and a decrease in tyrosine phosphorylation. Inhibition of PRDXs caused GAPDHS to undergo S-glutathionylation and the GSTO2 and SOD2 enzymes to undergo denitrosylation. Moreover, PRDX inhibition caused the AKAP proteins to be dephosphorylated.ConclusionsOur research provides evidence that crosstalk occurs between tyrosine phosphorylation and reversible oxPTMs during bull sperm capacitation. This study demonstrates that capacitation triggers S-nitrosylation and S-glutathionylation (and reverse reactions) of zona-pellucida binding proteins, which may be a new important mechanism that determines the interaction between sperms and oocytes. Moreover, TCA-related and flagellar proteins, which are particularly rich in PTMs, may play a key role in sperm capacitation. We propose that the deglutathionylation of ODFs and IZUMO4 proteins is a new hallmark of bull sperm capacitation. The obtained results indicate a relationship between PRDX activity and protein phosphorylation, S-glutathionylation and S-nitrosylation. The activity of PRDXs may be crucial for maintaining redox balance and for providing proper PKA-mediated protein phosphorylation during capacitation. Video Abstract.

Dataset Information

Posttranslational modifications of the bovine lens beaded filament proteins filensin and CP49.

Purpose

Methods

Results

Conclusions

Publications

Posttranslational modifications of the bovine lens beaded filament proteins filensin and CP49.

Similar Datasets

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