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. 2016 Aug:149:59-65.
doi: 10.1016/j.exer.2016.06.015. Epub 2016 Jun 23.

Spatial distributions of phosphorylated membrane proteins aquaporin 0 and MP20 across young and aged human lenses

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Spatial distributions of phosphorylated membrane proteins aquaporin 0 and MP20 across young and aged human lenses

Danielle B Gutierrez et al. Exp Eye Res. 2016 Aug.

Abstract

In the human ocular lens it is now realized that post-translational modifications can alter protein function and/or localization in fiber cells that no longer synthesize proteins. The specific sites of post-translational modification to the abundant ocular lens membrane proteins AQP0 and MP20 have been previously identified and their functional effects are emerging. To further understand how changes in protein function and/or localization induced by these modifications alter lens homeostasis, it is necessary to determine the spatial distributions of these modifications across the lens. In this study, a quantitative LC-MS approach was used to determine the spatial distributions of phosphorylated AQP0 and MP20 peptides from manually dissected, concentric layers of fiber cells from young and aged human lenses. The absolute amounts of phosphorylation were determined for AQP0 Ser235 and Ser229 and for MP20 Ser170 in fiber cells from the lens periphery to the lens center. Phosphorylation of AQP0 Ser229 represented a minor portion of the total phosphorylated AQP0. Changes in spatial distributions of phosphorylated APQ0 Ser235 and MP20 Ser170 correlated with regions of physiological interest in aged lenses, specifically, where barriers to water transport and extracellular diffusion form.

Keywords: Aquaporin 0; MP20; Mass spectrometry; Membrane protein; Ocular lens; Phosphorylation.

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Figures

Fig. 1
Fig. 1
Identification and quantitation of MP20 168–173 pSer170. MS/MS spectra from an 18 year old lens confirmed the identity of A) endogenous MP20 168–173 pSer170 and B) its isotopically labeled AQUA peptide internal standard. Within the MS/MS spectra, the asterisks represent loss of phosphoric acid. The peptide sequence and fragmentation are shown in the top left corner of each panel, where phosphorylation is indicated by “p” and the isotopically labeled amino acid is indicated by “l” (bottom panel only). The XICs of MP20 168–173 pSer170 (panel A, insert) and its AQUA internal standard (panel B, insert) show the difference in mass between the (M+2H)2+ ions for the endogenous and labeled peptides and the identical elution times of these peptides.
Fig. 2
Fig. 2
Distribution of MP20 Ser170 phosphorylation in various aged lenses. MP20 pSer170 is differentially distributed across the lens and becomes significantly lower in older lenses at a normalized lens distance of 0.81 (p = 0.02; vertical dotted line), remaining as such to the lens center. The value graphed here represents the amount of phosphorylated peptide measured per sample. The amount of phosphorylated peptide per picogram of lens membrane protein (as stated in the text) can be determined by dividing this value by 42,183 pg. R2 values for the curves are as follows, 18 yr–0.99, 19 yr–0.97, 23 yr–0.99, 51 yr–0.99, 54 yr–0.99, 60 yr–0.99. Error bars represent standard deviation. See Supplemental Fig. 4a for the 95% confidence bands of the curves.
Fig. 3
Fig. 3
Distribution of AQP0 Ser235 phosphorylation in various aged lenses. AQP0 pSer235 is differentially distributed, across the lens and becomes significantly lower in older lenses at a normalized lens distance of 0.71 (p = 0.04; vertical dotted line), remaining as such to the lens center. The value graphed here represents the amount of phosphorylated peptide measured per sample. The amount of phosphorylated peptide per picogram of lens membrane protein (as stated in the text) can be determined by dividing this value by 42,183 pg. R2 values for the curves are as follows, 18 yr–0.99, 19 yr–0.91, 23 yr–0.99, 51 yr–0.99, 54 yr–1.00, 60 yr–1.00. Error bars represent standard deviation. See Supplemental Fig. 4b for the 95% confidence bands of the curves.
Fig. 4
Fig. 4
Distribution of AQP0 Ser229 phosphorylation in various aged lenses. The average amount of AQP0 pS229 in young and aged lenses is not significantly different. The value graphed here represents the amount of phosphorylated peptide measured per sample. The amount of phosphorylated peptide per picogram of lens membrane protein (as stated in the text) can be determined by dividing this value by 42,183 pg. R2 values for the curves are as follows, 18 yr–0.99, 19 yr–0.93, 23 yr–0.99, 51 yr–0.93, 54 yr–1.00, 60 yr–0.99. Error bars represent standard deviation. See Supplemental Fig. 4c for the 95% confidence bands of the curves.
Fig. 5
Fig. 5
Comparison of phosphorylation relative to the permeability barrier. The maximum amount of phosphorylation pre-barrier and the minimum amount of phosphorylation post-barrier are shown for three peptides in both A) young and B) aged lenses. The asterisks represent a statistically significant (p < 0.05) difference between pre- and post-barrier amounts of phosphopeptide.
Fig. 6
Fig. 6
Potential effects of the differential distribution of AQP0 Ser235 phosphorylation. This model, based on Kalman et al., 2008, incorporates the present findings with data regarding the effect of phosphorylation on AQP0-Ca2+-CaM binding (Gold et al., 2012; Reichow and Gonen, 2008; Rose et al., 2008) and the effect of Ca2+-CaM on AQP0 water permeability (Kalman et al., 2008; Reichow and Gonen, 2008). A) In young lenses, phosphorylation of AQP0 Ser235 peaks at an average normalized lens distance of 0.82 (represented by the yellow shaded region (left) and remains fairly high, suggesting that young lenses will have efficient water flow throughout the lens, as indicated by the broad blue arrows. B) In aged lenses, phosphorylation of AQP0 Ser235 decreases significantly starting at a normalized lens distance of 0.71 (represented by the black shaded region, left), permitting Ca2+-CaM binding (purple horseshoe shape, right) and decreasing AQP0 water permeability, as indicated by the narrow blue arrows. This may play a role in the formation of a barrier to water transport in aged lenses. The design of the lens cartoon (panels A and B, left side) was derived from (Donaldson et al., 2001). Note, this is a cartoon and is meant to illustrate the described concept not specific molecular interactions.

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