Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells.
This study aims at understanding the molecular mechanisms that prevent an otherwise functional mutant of Trop2 (Q118E) to reach the plasma membrane. This mutation is associated with a rare ocular disease (gelatinous drop-like corneal dystrophy, GDLD). The data clearly demonstrate that inhibiting UGGT rescues in part the traffic block. The authors conclude that Trop2 is a substrate of UGGT and propose that drugs that inhibit these enzymes to be considered. In the treatment of disorders caused by transport-incompetent yet functional mutant proteins.
Although neither particularly ambitious in the aims nor providing unexpected results, this paper provides incremental insights into the UGGT quality control system. It also provides a possible mechanism for rescuing mild mutants linked to genetic diseases.
It is difficult to consider that the modulation of the UGGT/EDEM quality control mechanism could be a viable therapeutic strategy, regardless the future discovery of specific UGGT inhibitors. Moreover, the improvement observed for the Trop2-Q118E mutant analyzed is rather small (far from the level of control, and with structural alterations), and it is unclear how effective it could be for phenotype rescue.
The choice of a single, mild mutant has important downsides, especially considering that its rescue is partial. In terms of potential clinical applications. It would important to test other Tropp2 variants, and possibly different mutant glycoproteins. As discussed by the authors, the “rescue” provides mostly proteins that are not structurally native. It would be useful to expand this point in the Discussion. Which fraction of the protein is expected to be functional? Which could be the downsides of decreasing quality control fidelity, both for the protein of interest and the whole cell proteostasis? The authors should compare the rate of secretion of normal proteins and the fidelity of quality control of mutant proteins that lack N-glycans upon UGGT inhibition.
The authors may want to consider the following suggestions to reinforce the paper.
Figure 1A-B: The choice of two different cell lines, with two different approaches (live vs fixed cells) strengthens the results. Images of the same cell lines without plasmid transfection should be included to formally exclude autofluorescence.
Figure 1C-D: The results are clear and convincing. Yet, the authors should consider to include a co-staining of Trop2 with an ER marker.
Figure 2: While the result is clear, performing the multichannel intensity plot analysis on a single cell per condition (given the cell heterogeneity, the presence of bright intracellular granules for both WGA and Trop2, and the arbitrary positioning of the line for signal intensity plotting) reduces the informative value of the graphs. The authors should consider increasing the number of cells analyzed per condition.
Figure 3: In figure 3B, the three upper panels are slightly out of focus, and there are several bright dots in the WGA staining. Together, these factors reduce the clarity of the presence or absence of the protein of interest on the cell membrane. Better images should be obtained and shown.
Figure 4: Multiple bands are present in the different lanes (at least 3 in the WCL and at least 2 in the CRT-IP). The authors should determine or at least discuss their identity (different glycosylation forms?). Moreover, the different shapes of the WCL and IP lanes, makes the comparison difficult, even though the results are in support of the conclusions.
Figure 5: The explanation provided by the authors for this figure is not completely clear. In panel 5A, a different construct of the WT protein is used, but no clear explanation is provided. The differences between reduced and non-reduced lanes are difficult to spot, and not clearly highlighted. The amount of the protein of interest in the two conditions, moreover, is strikingly different, which makes the comparison very difficult. Panel 5B is clearer, even if the authors should better pinpoint the difference between the two extraction protocols (why did they do that? Which differences did they expect to see? Did they see them?). As the authors stress the concepts of retention, improved secretion and accumulation, it could be useful adding proteasomal inhibition (eg. MG132 treatment) to the conditions to compare.

The authors addressed most if not all the relevant points raised to the original file. This reviewer remains somewhat skeptical about the possibility of translating their interesting findings into therapeutics, but of course hoping to be prove wrong.