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Natarajan Perumal, Sebastian Funke, Norbert Pfeiffer, Franz Grus; Characterization and Post-Translational Modifications of Proline-Rich Protein 4 (PRR4) in Basal and Reflex Tear Proteome of Human. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2453. doi: https://doi.org/.
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It was previously reported that PRR4 is down-regulated in the tears of dry eye patients. This study was initiated considering the lack of comprehensive characteristics profile of PRR4 in tears of healthy subjects and, to understand the physiological roles of PRR4 in tears. Post-translational modifications and characterization of PRR4 in basal and reflex tears were determined in the study presented herein.
Tear samples were collected employing capillary method for basal and reflex tears. Proteomics approaches based on 1-DE and 2-DE combined with LC-ESI-LTQ-Orbitrap MS system were employed for characterization and determination of post-translational modifications of PRR4.
Identification and characterization of PRR4 in 2-DE utilizing LC-ESI-LTQ-Orbitrap MS yielded 10 PRR4 spots with vast polymorphisms (molecular weight: 17 - 30 kDa, pI: 3.5 - 6.6). A new isoform designated as PRR4γ was also identified in addition to the two existing isoforms of PRR4. Further characterization of the PRR4 isoforms identified multiple post-translational modifications, which are methylation, oxidation and pyroglutamate formation. Label-free quantification analysis of basal and reflex tear proteome employing 1-DE and LC-ESI-LTQ-Orbitrap MS demonstrated significant increment of PRR4 for the first time in reflex tears. Correspondingly, the result also showed that more methylation, oxidation and pyroglutamate formation were found in reflex tears than in basal tears. Besides, reflex tear sample also showed significant increment of prolactin, serum albumin, zinc-α-2-glycoprotein, haptoglobin, mesothelin and β-2-microglobulin and, decrement of Ig α-1 chain C, cystatin-S, clusterin, lipid transfer protein II and phospholipase A2 compared to basal tears.
The results of this study provide fundamental insight into the complex characteristics of PRR4 in basal and reflex tears. These findings highlight that the multiple post-translational modifications of PRR4 isoforms may play crucial roles in the tear system and ocular surface of healthy people as well as in dry eye patients.
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