Deamidation delays expression of βB2-crystallin in the zebrafish lens

Abstract

Purpose : The role of the major protein modification, deamidation, was investigated in the lens by overexpressing the deamidated βB2-crystallin in the zebrafish.

Methods : Zebrafish embryos at the single-cell stage were microinjected with DNA plasmid constructs containing a zebrafish lens αA crystallin promoter, the human βB2 crystallin gene, and a yellow fluorescent protein tag (YFP). The construct also codes for green fluorescent protein (GFP) expressed in the heart for visual screening of successful transgenesis. The final expression plasmid was injected into the embryo at the single-cell stage along with capped transposase RNA for gene integration. Fish displaying GFP and/or YFP were back-crossed and bred with wild-type fish to generate an F₁ generation and then crossbred to generate F₂ offspring for a stable germ line. We have generated zebrafish expressing wildtype βB2 (WT) and the deamidated mutant (DM), Q70E/Q162E βB2.

Results : We confirmed expression of our mutant protein with high-resolution mass spectrometry. We digested lenses from F₁ and F₂ WT zebrafish spiked with a known amount of ¹⁵N-labeled βB2 and determined the ratio of light to heavy peptides. We estimate there are 3.5 and 6 ng of expressed human βB2 in 10 ug of lens digests from F₁ and F₂ fish, respectively. Since, there is a single tryptic peptide shared between zebrafish and human βB2, we were also able to estimate the amount of native βB2 to be 0.8 ug in 10 ug of lens digest. Even though the expressed human βB2 is more than a 100-fold less than the native βB2, significant fluorescence was observed throughout the lens. Fluorescence in the lens was detected in >75% of the F₁ and F₂ offspring of WT with fluorescence in the heart at 5 dpf. We also confirmed expression of DM βB2. Only 15-50% of F₁ offspring of DM zebrafish had fluorescence in the lens, even though fluorescence in the heart was present. When these zebrafish were screened again after 2-3 months, fluorescence was detected in lenses of more zebrafish. Furthermore, attempts to generate F₂ offspring of DM zebrafish proved difficult.

Conclusions : Expression of WT and DM βB2 in the zebrafish lens does not alter lens opacity in the young zebrafish. However, deamidation delays expression of βB2 in the lens and may affect development. Zebrafish are being monitored to determine the effects of deamidation in the older zebrafish.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.