Lens crystallin modifications and cataract in transgenic mice overexpressing acylpeptide hydrolase

Abstract

The accumulation of crystallin fragments in vivo and their subsequent interaction with crystallins are responsible, in part, for protein aggregation in cataracts. Transgenic mice overexpressing acylpeptide hydrolase (APH) specifically in the lens were prepared to test the role of protease in the generation and accumulation of peptides. Cataract development was seen at various postnatal days in the majority of mice expressing active APH (wt-APH). Cataract onset and severity of the cataracts correlated with the APH protein levels. Lens opacity occurred when APH protein levels were >2.6% of the total lens protein and the specific activity, assayed using Ac-Ala-p-nitroanilide substrate, was >1 unit. Transgenic mice carrying inactive APH (mt-APH) did not develop cataract. Cataract development also correlated with N-terminal cleavage of the APH to generate a 57-kDa protein, along with an increased accumulation of low molecular weight (LMW) peptides, similar to those found in aging human and cataract lenses. Nontransgenic mouse lens proteins incubated with purified wt-APH in vitro resulted in a >20% increase in LMW peptides. Crystallin modifications and cleavage were quite dramatic in transgenic mouse lenses with mature cataract. Affected lenses showed capsule rupture at the posterior pole, with expulsion of the lens nucleus and degenerating fiber cells. Our study suggests that the cleaved APH fragment might exert catalytic activity against crystallins, resulting in the accumulation of distinct LMW peptides that promote protein aggregation in lenses expressing wt-APH. The APH transgenic model we developed will enable in vivo testing of the roles of crystallin fragments in protein aggregation.

Keywords: Acylpeptide Hydrolase; Cataract; Crystallins; Lens; Peptides; Protease; Protein Degradation; Transgenic Mice.

FIGURE 1.

Construction of transgenic mice expressing APH specifically in the lens. A, schematic diagram of transgenic construct. Sus scropa APH cDNA (2451 bp) containing an N-terminal T7 tag and C-terminal His tag was inserted between a modified αA promoter. B, appearance of a 30-day-old lens expressing active APH (KKS4) and inactive APH (KKS4M).

FIGURE 2.

Characteristics of transgenic mice. A, specific activity of APH in 21-day-old transgenic mouse lenses. The activity was measured kinetically for 5 min using 100 nmol of Ac-Ala-pNA substrate. The activities are average ± S.E. from five lenses estimated individually. Activities of APH in lenses expressing active enzyme were significantly higher than those of mutant and nontransgenic controls (p < 0.0001). Mice expressing mt-APH did not have measurable activity, which was comparable with nontransgenic controls. B, time required for cataract development in various transgenic lines.

FIGURE 3.

Appearance of lenses from various transgenic mouse lines expressing wild-type and mutant APH at different postnatal periods. d, days.

FIGURE 4.

SDS-PAGE analysis of transgenic lenses. Lenses from 30-day-old mice were homogenized in urea buffer, and the supernatant containing 150 μg of the protein was run on the gel (1–7 lanes from the left). The arrow points to the APH band. The amount of APH protein expressed in different transgenic lines was estimated from the image and is given in parentheses on top of the lane as a percentage of total protein. The protein profiles of the transparent and cataract region of a lens expressing wt-APH are given in the right next to the Marker lane. Strong APH band was seen in the transparent region, suggesting that the cataract aggregate is not due to precipitation of APH protein.

FIGURE 5.

Western blot analysis of transgenic lenses. A, comparison of protein levels between nontransgenic (−) and transgenic (KKS4) (+) lenses at various postnatal periods. A small fraction of APH was cleaved, resulting in a 57-kDa fragment (arrow). β- and γd-crystallin levels were lower in transgenic lenses at all assay periods. B, Western blot of 30-day-old lenses from different transgenic lines. The fragmented 57-kDa APH (arrow) was seen only in lenses that are certain to develop cataract.

FIGURE 6.

A–F, H&E staining of transgenic mice lens sections at various postnatal time periods. A, postnatal day 7 (KKS2) showing normal lens formation. B, postnatal day 42 (KKS4) showing expelled primary fiber cells. The square regions are enlarged to show the rupture of the posterior capsule and vacuole in the epithelial cell. C, postnatal day 90 (KKS2) showing disintegrating fiber cells and large vacuoles in lens. D, postnatal day 50 (KKS1M) showing normal lens development in lens expressing inactive APH. The square region is enlarged to show no vacuole formation in epithelial cell. E, postnatal day 90 (KKS3M) showing no apparent lens abnormalities. F, postnatal day 90 nontransgenic lens. G–L, immunohistochemistry of APH in transgenic lens sections at various postnatal time periods. G, postnatal day 7 KKS2 eye section showing uniform expression of APH (brown) in the lens. H, postnatal day 60 KKS2 eye showing vacuoles in the lens epithelium and fiber cells with intense staining for APH in the cortical region. I, postnatal day 90 KKS2 lens. J, postnatal day 7 KKS5 lens showing moderate APH expression. K, postnatal day 90 KKS1M lens. L, nontransgenic lens at postnatal day 7.

FIGURE 7.

MALDI-TOF-MS analysis of LMW peptides in 30-day postnatal lenses of transgenic mice expressing inactive APH (mt-APH) and active APH (wt-APH). The mass ions in pink are distinct to the 30-day-postnatal lenses. The mass ions in cyan were present in both groups, but the intensity varied significantly. The mass ions in black are common to both groups. The sequences identified by Nanospray QqTOF MS/MS analysis are given in parentheses. (*, Intensity was truncated for scaling purpose.)

FIGURE 8.

The two-dimensional DIGE analysis of 90-day-old nontransgenic and transgenic mice expressing active APH. A, two-dimensional gel of whole lens extract labeled with Cy3 (nontransgenic) and Cy5 (transgenic) dyes. The image was scanned and analyzed using Decyder software. The gel was stained with Coomassie Blue, and the protein spots that were picked for mass spectrometry are shown as dotted circles. See Table 1 for the identity of proteins present in each spots picked from A. B, spot 226 (area marked in pink) from two-dimensional DIGE gel was identified as APH by mass spectrometry. C, spot 375 (area marked in pink) from two-dimensional DIGE gel was identified as truncated APH. The areas marked in blue and red are other spots selected by the software to estimate the relative fold difference in protein levels between transgenic and nontransgenic lenses.

FIGURE 9.

MALDI-TOF-MS analysis of LMW peptides from the in vitro incubation of mouse lens proteins with wt-APH. Nontransgenic mouse lens proteins (10 mg) were incubated without (A) or with purified wt-APH (B and C) and in the absence (A and C) and presence (B) of protease inhibitor at 37 °C for 24 h. Incubation with active APH (C) resulted in a >20% increase in LMW peptides compared with control groups. The mass ion corresponding to that in pink was also present in vivo (Fig. 7). The mass ion in red was 8–10-fold more abundant in the active APH incubations. *, intensity truncated at 5000.