Abstract
Purpose :
FN3K is a natural intracellular repair enzyme for condensation products of glucose with protein primary amine groups. The enzymes action on AGEs, high molecular weight compounds enhancing oxidative stress and covalent crosslinking, is yet unknown. We produced active recombinant FN3K enzyme (rFN3K) and inactive mutant FN3K (mFN3K) in yeast (Pichia pastoris) and investigated the activity of rFN3K against AGEs in human lenses.
Methods :
A DNA construct encoding human FN3K flanked by a N-terminal His-tag with a caspase-cleavable D–E–V–D site was codon-optimized for Pichia pastoris and cloned into the pKai61 vector for intracellular expression. For mFN3K, human FN3K carrying a K41L substitution (FN3K–K41L) in the putative F–V–K catalytic triad was made. After gel filtration, kinase activity (or lack thereof for mFN3K) was tested in vitro. The protein was further characterized on SDS-PAGE and Western blot. Human lens material obtained after phacoemulsification (n=20), was incubated for 3 h with rFN3K or mFN3K. AGEs were measured by Maillard-type autofluorescence (AF) (excitation 365 nm, emission 390–700 nm). Fructose containing AGEs were investigated in the urea-soluble fraction of rFN3K treated human lens material by gel filtration. Individual fractions were photometrically tested (488 nm) by Seliwanoff reaction.
Results :
Remarkable decreases of AF were observed for rFN3K compared to mFN3K. Pronounced decrease of AF was found after 1 h incubation (43.6–49.7% decrease compared to baseline). Additional decrease of AF was found after 2 h incubation (56.7–70.8% decrease compared to baseline), and remained stable thereafter. At lower concentration range of rFN3K (12.5 µg/mL, 6.25 µg/mL and 1.25 µg/mL) a more notable time- and dose dependent effect was seen. Gel filtration showed breakdown of fructose-containing AGEs in human lens fragments by rFN3K. Before treatment, presence of AGEs was observed by two peaks with molecular mass of 2500 Da and 1660 Da. After rFN3K treatment, breakdown products with molecular mass ranging between 1500 and 2500 Da were detected.
Conclusions :
rFN3K and mFN3K enzyme were successfully produced in Pichia pastoris. AF kinetics on human lens material revealed a dose and time dependent effect of treatment with rFN3K. A breakdown of high molecular mass fructose related AGEs by rFN3K was observed. Further research is needed to identify the exact substrates of rFN3K in human lenses.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.