Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells

Abstract

Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ERlocalised 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 droplike 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 rescueof-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.

Keywords: GDLD; TACSTD2; Trop-2; UGGT; UGGT1; UGGT2; glycoprotein secretion; responsive mutant.

Figure 1.. Rescue of secretion of the Trop-2-Q118E-EYFP- mutant glycoprotein upon UGGT1 inactivation.

Confocal images of single optical sections of transiently transfected HEK 293T (live imaging) and Vero E6 cells (fixed cells). Nuclei were stained with Hoechst 33342 in HEK 293T cells or with DAPI stain in Vero E6 cells (blue). (A, B) Wild type (WT) and UGGT1^−/− HEK 293T and Vero E6 cells were transiently transfected with Trop-2-pEYFP-N1 and Trop-2-Q118E-pEYFP-N1 plasmids. (A) The Trop-2-EYFP wild-type glycoprotein (green) traverses the secretory pathway and reaches the plasma membrane (white arrowheads); (B) in WT cells, the Trop-2-Q118E-EYFP mutant glycoprotein (green) is visible in the secretory pathway but absent from the plasma membrane. In the UGGT1^−/− cells, the same Trop-2-Q118E-EYFP mutant glycoprotein reaches the cellular membrane (white arrowheads). (C, D) WT, UGGT1^−/−, UGGT2^−/−, and UGGT1/2^−/− HEK 293T cells were transiently transfected with Trop-2-pEYFP-N1 (C) and Trop-2-Q118E-pEYFP-N1 (D) plasmids. Wheat Germ Agglutinin (WGA) Alexa Fluor 555 (orange) enables verification of plasma membrane localisation of the fluorescent fusion glycoproteins; (C) in both WT and UGGT1^−/− HEK 293T cells the Trop-2-EYFP-WT glycoprotein (green) reaches the cellular membrane (white arrowheads); (D) in the WT HEK 293T cells the Trop-2-Q118E-EYFP mutant glycoprotein (green) is trapped in the secretory pathway, while in the UGGT1^−/− and UGGT1/2^−/− HEK 293T cells (and to a lower extent in UGGT2^−/− cells) the same mutant glycoprotein is visible both in the ER and in the membrane (white arrowheads).

Figure 2.. Multichannel intensity plots of the Trop-2-pEYFP-N1 transfected HEK 293T cell lines.

For each combination of cell line and transiently transfected vector, the fluorescent signal is plotted along the line drawn in white in the inset panel. Labelling: green = EYFP; orange = WGA Alexa Fluor 555; blue = Hoechst 33342. The signal from the WGA Alexa Fluor 555 labelled membrane (orange) overlaps with EYFP (green) signals in Trop-2-pEYFP-N1 transfected cells (black arrowheads, top row). In case of the mutant Trop-2-Q118E-pEYFPN1 transfected cells, the protein is absent from the membrane in WT cells (bottom left panel). The deletion of one or both UGGT genes rescues secretion of the mis-folded Trop-2 mutant glycoprotein (black arrowheads, all bottom panels but the leftmost one).

Figure 3.. The Trop-2-Q118E-EYFP lack of secretion phenotype is restored in HEK 293T UGGT1^−/− cells re-complemented with the UGGT1 gene.

(A) UGGT1 and UGGT1 D1454A re-complementation does not interfere with the localisation of the WT Trop-2-EYFP glycoprotein (green): the protein traverses the secretory pathway and reaches the plasma membrane (white arrowheads); (B) In re-complemented WT cells, the Trop-2-Q118E-EYFP mutant glycoprotein (green) is absent from the plasma membrane and is retained in the secretory pathway (top first and second rows). In the UGGT1 re-complemented UGGT1^−/− cells, we observe restoration of the original lack of secretion phenotype observed for Trop-2Q118E-EYFP in WT cells: the Trop-2-Q118E-EYFP mutant glycoprotein does not reach the cellular membrane (third row). Consistently with these observations, we still observe the rescue of secretion of the mutant glycoprotein in UGGT1^−/− cells recomplemented with the inactive UGGT1 D1454A mutant (bottom row, white arrowheads). (C) Multichannel intensity plots of the Trop-2-EYFP transfected recomplemented cell lines confirm our visual observations (black arrowheads).

Figure 4:. The in cellula glucosylation by UGGT1 of Trop-2-EYFP (WT and Q118E mutant).

(A) The synthesis and modification of N-linked glycans starting from the dolichol-PP-Man₉GlcNAc₂ stage within the ER lumen. ALG6 appends the first glucose onto the immature glycan using dolichol-P-glucose as the source. The mono-glucosylated glycan is then built into a tri-glucosylated carbohydrate by ALG8/10 prior to covalent linking to the nascent peptide chains. The glycan is trimmed to a mono-glucosylated state by glucosidases I and II, whereupon it can bind ER lectin chaperones. Removal of the remaining glucose by glucosidase II dissociates the glycoprotein from the lectin chaperones, whereby three options are possible: continuation of trafficking, degradation, or reglucosylation by the UGGTs. Orange ovals represent dolichol anchors, yellow circles phosphates, blue squares GlcNAc, green circles mannose, and blue circles glucose. (B) Alteration of glycan synthesis pathway for glucosylation studies. Deletion of ALG6 results in a Man₉GlcNAc₂ glycan being transferred to nascent chains by the OST as the initial glucose can no longer be added. Interaction with ER lectin chaperones is thereby facilitated solely through glucosylation by the UGGTs. Further enrichment is achieved through treatment with DNJ, preventing the removal of the inner glucose. (C) The designated cell lines were transfected with either the WT Trop-2-EYFP or Q118E mutant and lysed. The lysate was split between a whole cell lysate sample (20%) and a GST-CRT (35%) and GST-CRT-Y109A pulldown (35%) and resolved by 9% SDSPAGE before transferring to a PVDF membrane. Imaged is an αEYFP immunoblot. Data represent three independent biological replicates. (D) Mock transfection to measure background binding of αEYFP antibody against whole cell lysate from each cell line. The membrane was also probed against GAPDH as a loading control. (E) Quantification of blots from (B). Percent glucosylation was calculated by subtracting the amount of protein pulled down in the Y109A lane from the CRT for each cell line. The resulting value was divided by the normalised quantified protein within the whole cell lysate and multiplied by 100. Error bars represent the standard deviation. **** represents P ≤ 0.0001 and *** represents P ≤ 0.001.

Figure 5.. The Q118E mutation generates inter-molecularly SS-bonded mis-folded Trop-2-Q118E-pEYFP-N1 multimers

(A) ) Anti-EYFP Western blot of membrane proteins isolated by Lipodisq extraction 96 hrs post-transfection from HEK 293T UGGT1^−/− cells transiently transfected with Trop-2-TEV-GFP-6xHis WT and the Trop-2-Q118E mutant in the presence or absence of TCEP. (B) Anti-EYFP Western blot of membrane fractions extracted with the CelLytic^™ MEM Protein Extraction Kit from HEK 293T cells transiently transfected with the Trop-2-pEYFP-N1 (“WT”) and Trop-2-Q118E-pEYFP-N1 plasmids (“M”). Samples were run in the abeance or presence of reducing agent..

Figure 6.. Assessment of the secretion-rescued Trop-2 folding.

(A) Summary of the gating and signal acquisition strategy. (B) Left panels: dot plots of the experiments showing dimensional parameters (forward scatter, FSC, and side scatter, SSC). UGGT1^−/− Vero E6 cell singlets (R1) were gated out for subsequent analysis of UGGT1^−/− Vero E6 cells expressing WT or Q118E-Trop-2-EYFP proteins. Middle panels: Fluorescent signals in the FITC/YFP channel relative to the R1 cells are shown. YFP⁺ cells were gated out for following analysis. Right panels: histogram plots showing the expression levels of membranous Trop-2 as measured by staining with an anti Trop-2 pAb (blue) or mAb (red). Ratios between mAb and pAb Mean Fluorescence Intensities are shown for each experimental group.