Rescue of secretion of rare‐disease‐associated misfolded mutant glycoproteins in UGGT1 knock‐out mammalian cells

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER‐localized eukaryotic glycoprotein secretion checkpoint, UDP‐glucose glycoprotein glucosyl‐transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re‐glucosylating 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”). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop‐2‐Q118E, E227K and L186P mutants, which cause gelatinous drop‐like corneal dystrophy (GDLD). Compared with the wild‐type Trop‐2, which is correctly localized at the plasma membrane, these Trop‐2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop‐2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9‐mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop‐2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1−/−cells. UGGT1 also efficiently reglucosylated Trop‐2‐Q118E‐EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad‐spectrum rescue‐of‐secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. “The figure depicts the transformative impact of UGGT1 gene deletion in mammalian cells on the intracellular fate of fluorescent chimeras representing human Trop‐2 Q118E, E227K and L186P glycoprotein mutants, implicated in gelatinous drop‐like corneal dystrophy. In wild‐type cells (UGGT1+/+), the mutants are ensnared within the endoplasmic reticulum (ER), symbolized by the trapped green protein. Conversely, in UGGT1 knockout cells (UGGT1/−), the green protein successfully navigates to the cell membrane, illustrating the rescue of secretion. Notably, the Trop‐2‐Q118E glycoprotein, a disease mutant, undergoes efficient glucosylation by UGGT1 in human cells, establishing its classification as a genuine cellular UGGT1 substrate.