Growth and Antioxidant-Related Effects of the Reestablished Ascorbic Acid Pathway in Zebrafish (Danio rerio) by Genomic Integration of L-Gulonolactone Oxidase From Cloudy Catshark (Scyliorhinus torazame).

K A S N Shanaka,J R P Jayasingha,Jehee Lee,Myoung-Jin Kim,N D Janson,K P Madushani,Sumi Jung

doi:10.3389/fphys.2021.685595

Abstract

Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.

Highlights

L-gulonolactone oxidase (GULO) is the last enzyme in the ascorbic acid (AA or vitamin C) pathway
Electron carriers could be weakly expressed in the presence of AA and could explain the reduced expression of the electron carrier cyb5a in Tg fish compared with Wt fish
The catalytic activity of sGULO in Tg zebrafish was confirmed by analyzing the AA production

Summary

Introduction

L-gulonolactone oxidase (GULO) is the last enzyme in the ascorbic acid (AA or vitamin C) pathway. Almost all plants and a majority of animals have an active GULO enzyme that catalyzes AA synthesis from glucose (Smirnoff, 2001). Prokaryotes lack this ability (Smirnoff, 2001), and obtaining AA through symbiotic relationships from prokaryotes is not an option for the animals. Animals who do not have an active GULO enzyme must obtain AA from their diet. AA has a regulatory role during development (Gallie, 2013) and reported immune defense functions in animals (Carr and Maggini, 2017)

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