| Autor: |
Gabor Tax, Kevin P Guay, Tatiana Solde, Charlie J Hitchman, Johan C Hill, Snezana Vasilievic, Andrea Lia, Carlos P Modenutti, Kornelis R Straatman, Maurizio Molinari, Angelo Santino, Daniel N Hebert, Nicole Zitzmann, Pietro Roversi, Marco Trerotola |
| Rok vydání: |
2023 |
| Předmět: |
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| Zdroj: |
bioRxiv |
| DOI: |
10.1101/2023.05.30.542711 |
| Popis: |
Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ER- localised eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognises a mis-folded glycoprotein and flags it for ER retention by reglucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease even if the mutant glycoprotein retains activity (“responsive mutant”). Here, we investigated the subcellular localisation of the human Trop-2 Q118E variant, which causes gelatinous drop- like corneal dystrophy (GDLD). Compared with the wild type Trop-2, which is correctly localised at the plasma membrane, the Trop-2-Q118E variant is found to be heavily retained in the ER. Using Trop-2-Q118E, we tested UGGT modulation as a rescue-of-secretion therapeutic strategy for congenital rare disease caused by responsive mutations in genes encoding secreted glycoproteins. We investigated secretion of a EYFP-fusion of Trop-2-Q118E by confocal laser scanning microscopy. As a limiting case of UGGT inhibition, mammalian cells harbouring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 gene expressions were used. The membrane localisation of the Trop-2-Q118E-EYFP mutant was successfully rescued in UGGT1 (-/-) and UGGT1/2 (-/-) cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula . The study supports the hypothesis that UGGT1 modulation constitutes a novel therapeutic strategy for the treatment of Trop-2-Q118E associated GDLD, and it encourages the testing of modulators of ER glycoprotein folding Quality Control (ERQC) as broad-spectrum rescue- of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. SYNOPSIS: Deletion of the UGGT1 and UGGT1/2 genes in HEK 293T cells rescues secretion of an EYFP-fusion of the human Trop-2-Q118E glycoprotein mutant. The mutant is retained in the secretory pathway in wild type cells and it localises to the cell membrane in UGGT1 (-/-) single and UGGT1/2 (-/-) double knock-out cells. The Trop-2-Q118E glycoprotein disease mutant is efficiently glucosylated by UGGT1 in human cells demonstrating that it is a bona fide cellular UGGT1 substrate. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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