Deletion of Seventeen Amino Acids at the C-Terminal End of Aquaporin 0 Causes Distortion Aberration and Cataract in the Lenses of AQP0ΔC/ΔC Mice

Abstract

Purpose: Investigate the effects of the absence of 17 amino acids at the C-terminal end of Aquaporin 0 (AQP0) on lens transparency, focusing property, and homeostasis.

Methods: A knockin (KI) mouse model (AQP0ΔC/ΔC) was developed to express AQP0 only as the end-cleaved form in the lens. For this, AQP0 was genetically engineered as C-terminally end-cleaved with amino acids 1 to 246, instead of the full length 1 to 263 of the wild type (WT). After verifying the KI integration into the genome and its expression, the mouse model was bred for several generations. AQP0 KI homozygous (AQP0ΔC/ΔC) and heterozygous (AQP0+/ΔC) lenses were imaged and analyzed at different developmental stages for transparency. Correspondingly, aberrations in the lens were characterized using the standard metal grid focusing method. Data were compared with age-matched WT, AQP0 knockout (AQP0-/-), and AQP0 heterozygous (AQP0+/-) lenses.

Results: AQP0ΔC/ΔC lenses were transparent throughout the embryonic development and until postnatal day 15 (P15) in contrast to age-matched AQP0-/- lenses, which developed cataract at embryonic stage itself. However, there was distortion aberration in AQP0ΔC/ΔC lens at P5; after P15, cataract began to develop and progressed faster surpassing that of age-matched AQP0-/- lenses. AQP0+/ΔC lenses were transparent even at the age of 1 year in contrast to AQP0+/- lenses; however, there was distortion aberration starting at P15.

Conclusions: A specific distribution profile of intact and end-cleaved AQP0 from the outer cortex to the inner nucleus is required in the lens for establishing refractive index gradient to enable proper focusing without aberrations and for maintaining transparency.

Figure 1

Strategy to generate a AQP0^ΔC/ΔC by introducing a stop codon after amino acid 246. (A) WT: Schematic structure of WT mouse AQP0 gene showing exons 1-4 (as rectangular vertical or horizontal boxes) and the connecting introns. Vector: Exons 3 and 4 with introns (highlighted in blue and red) as well as a Neo selection gene were amplified by PCR and cloned into the vector (details in [B]). Black dotted lines on either side denote vector sequences. Asterisk indicates an in-frame translation stop codon predicted to truncate AQP0 after the amino acid Asparagine-246. KI-Neo: The recombinant vector (with Exons 3 and 4, introns highlighted in blue and red and the Neo selection gene) was transfected into mouse embryonic stem cells and positive clones were selected using the Neo selection marker. KI: KI with stop codon after amino acid 246 but without the Neo-Selection marker. Light green vertical rectangle indicates one set of the LoxP-FRT sites that remain after Neo deletion (74 bp). Positive embryonic stem cells selected were injected into mouse blastocysts to develop AQP0 KI mouse model (AQP0^ΔC/ΔC). (B) Schematic of the introduction of the stop codon through a point mutation incorporated into a primer pair (to delete the 17 amino acids after the 246^th), and the KI targeting vector design. The C-terminal end deletion was engineered by overlap extension PCR using the primers as indicated. Red rectangle represents the deletion region. SA, short homology arm; LA, long homology arm. N1, N2, PT2, PT3, PT4, P6, and T73 are forward or reverse primers used, as indicated.

Figure 2

Screening for the introduced stop codon. (A) A 676-bp fragment was amplified using revneo3b/MOPE4 primers. MOPE4 is located on the long homology arm (LA, see Fig. 1B), downstream of the deletion. Revneo3b is located inside the Neo cassette. Among the eight agouti-colored chimeras tested, three (#889, 890, and 893) were positive. The PCR product amplified by primers revneo3b and MOPE4 was sequenced to confirm the presence of the introduced stop codon and the fidelity of the amplified fragment. (B) Screening for Neo deletion. A primer set Neo deletion primer 1 (NDEL1) and NDEL2 was used to screen mice for the deletion of the Neo cassette. The PCR product for the wild-type is 233 bp. A second band with 307 bp indicates Neo deletion in the chimeras (after Neo deletion, one set of LoxP-FRT sites remain [74 bp] in the KI chimeras). (C) Screening for presence of FLP transgene. A primer set, FLP1 and FLP2, used to screen mice for presence of the FLP transgene amplified a positive product of 725 bp; (D) FLP-positive and Neo-negative mice were crossed with C57 WT to eliminate FLP transgene. KI mice #889, 890, and 893 were FLP negative with no amplification product while FLP-positive mice genomic DNA amplified 725-bp product. MW, molecular weight marker.

Figure 3

(A) Genotyping of C57 WT and KI (AQP0^+/ΔC and AQP0^ΔC/ΔC), and FVB WT mice to show the absence or presence of CP49 natural mutation. An amplicon of 320 bp indicates the presence of intact CP49 allele; 386-bp amplicon indicates the presence of mutant CP49 allele. (B) Western blotting of lens proteins of C57 WT and KI mice (AQP0^+/ΔC and AQP0^ΔC/ΔC) to confirm the expression of CP49 protein. Blot treated with CP49 antibody (arrow CP49, ∼49 kDa).

Figure 4

(A) Protein expression profile using MALDI/TOF/MS. Lenses of mouse pups at P5 from WT, AQP0^+/ΔC, and AQP0^ΔC/ΔC were used. (B) (Top) Western blotting of P5 lens proteins of WT (lane 1), AQP0^+/ΔC (lane 2), and AQP0^ΔC/ΔC (lane 3). The bar graph on the right shows quantification of the immunoreactive protein bands of AQP0^+/Δ by densitometry. Values from five independent immunoblots of AQP0^+/ΔC lens membrane proteins were used. Levels of AQP0 C-terminal antibody binding were not significantly (P > 0.05) different between the immunoreactive bands of intact AQP0 and C-terminal end-cleaved AQP0. (Bottom) Immunostaining. P5 lens sagittal sections of WT and AQP0^ΔC/ΔC at low magnification. Fluorescent signals in the whole-lens sections appear nonuniform at different areas under low magnification both in the WT and KI lenses due to the thickness of the cryosections, which does not permit uniformity in the imaging plane. White bar, 150 μm. Lens cross sections of WT and AQP0^ΔC/ΔC are shown below at high magnification. Mutant AQP0 protein localized in the fiber cell membranes as in the case of WT lens. Yellow bar, 4 μm. The bar graph on the right shows quantification of the fluorescence intensity from four independent immunostainings of lens cross sections of WT and AQP0^ΔC/ΔC mice. Level of fluorescence due to AQP0 C-terminal antibody binding was not significantly different (P > 0.05) between WT and AQP0^ΔC/ΔC lens sections.

Figure 5

Qualitative characterization of transparency (left column) and aberration (right column) of WT, AQP0^−/−, and AQP0^ΔC/ΔC mouse lenses at (A) E18 (white bar: 200 μm), (B) P5 (bar: 320 μm), (C) P10 (bar: 350 μm), (D) P15 (bar: 400 μm), and (E) P20 (bar: 425 μm). (F) Quantification of pixel brightness intensity to assess lens transparency in WT, AQP0^−/−, and AQP0^ΔC/ΔC mouse lenses at E18, P5, P10, P15, and P20. Higher the pixel brightness intensity, lower would be the lens transparency. Star indicates statistical significance, AQP0^ΔC/ΔC compared with WT at P20 (P < 0.001); filled triangle indicates statistical significance, AQP0^ΔC/ΔC compared with AQP0^−/− (P < 0.05). Note that at P20 lens transparency of AQP0^ΔC/ΔC lens is much less than that of AQP0^−/− lens.

Figure 6

Qualitative characterization of transparency (left column) and aberration (right column) of WT, AQP0^+/−, and AQP0^+/ΔC mouse lenses. (A) P15 (white bar: 400 μm), (B) 2-month-old (bar: 700 μm; white circle denotes the area of aberration), and (C) 12-month-old (bar: 800 μm; white circle denotes the area of aberration). (D) Quantification of pixel brightness intensity in WT, AQP0^+/−, and AQP0^+/ΔC mouse lenses at P15, 2, and 12 months of age. Higher the pixel brightness intensity, lower would be the lens transparency. Star indicates statistical significance, AQP0^+/ΔC compared with WT (P < 0.001); open triangle indicates statistical significance, AQP0^+/ΔC compared with AQP0^+/− (P < 0.05). Note that lens transparency of AQP0^+/ΔC lens is higher than that of AQP0^+/− lens.

Figure 7

Distortion aberration zones in the lenses of AQP0^ΔC/ΔC mouse that expresses only C-terminal end-cleaved AQP0. (A) An early stage in lens development (P5) showing two distortion aberration zones. (B) A later stage (P15) showing three distortion aberration zones.

Figure 8

Western blot analysis. Membrane proteins extracted from MDCK cells transfected with mouse intact AQP0 or from the lenses of P10 and 4-month-old adult C57 WT mice were tested. AQP0 C-terminal-specific antibody was used. The C-terminal antibody was raised using an epitope peptide having amino acids 220 to 263. Of note, here, the KI-truncated AQP0 has amino acids up to 246 at the C-terminus.