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

Autor:	Tax G; Leicester Institute of Chemical and Structural Biology and Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 7HR, England, United Kingdom., Guay KP; Department of Biochemistry and Molecular Biology, and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, United States., Soldà T; Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana (USI), Bellinzona, Switzerland., Hitchman CJ; Leicester Institute of Chemical and Structural Biology and Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 7HR, England, United Kingdom., Hill JC; Institute of Glycobiology, Department of Biochemistry, South Parks Road, Oxford OX1 3RQ, United Kingdom., Vasiljević S; Institute of Glycobiology, Department of Biochemistry, South Parks Road, Oxford OX1 3RQ, United Kingdom., Lia A; Leicester Institute of Chemical and Structural Biology and Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 7HR, England, United Kingdom.; Institute of Sciences of Food Production, ISPA-CNR Unit of Lecce, via Monteroni, I-73100 Lecce, Italy., Modenutti CP; Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (FCEyN-UBA) e Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN) CONICET, Pabellón 2 de Ciudad Universitaria, Ciudad de Buenos Aires C1428EHA, Argentina., Straatman KR; Leicester Institute of Chemical and Structural Biology and Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 7HR, England, United Kingdom.; Core Biotechnology Services, University of Leicester, University Road, Leicester LE1 7RH, England, United Kingdom., Santino A; Institute of Sciences of Food Production, ISPA-CNR Unit of Lecce, via Monteroni, I-73100 Lecce, Italy., Molinari M; Institute of Glycobiology, Department of Biochemistry, South Parks Road, Oxford OX1 3RQ, United Kingdom.; School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland., Zitzmann N; Institute of Glycobiology, Department of Biochemistry, South Parks Road, Oxford OX1 3RQ, United Kingdom., Hebert DN; Department of Biochemistry and Molecular Biology, and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, United States., Roversi P; Leicester Institute of Chemical and Structural Biology and Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 7HR, England, United Kingdom.; Institute of Agricultural Biology and Biotecnology, IBBACNR Unit of Milano, via Bassini 15, I-20133 Milano, Italy., Trerotola M; Department of Medical, Oral and Biotechnological Sciences, 'G. d'Annunzio' University of Chieti-Pescara, Italy; Laboratory of Cancer Pathology, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti-Pescara, Italy.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 May 31. Date of Electronic Publication: 2023 May 31.
DOI:	10.1101/2023.05.30.542711
Abstrakt:	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.
Databáze:	MEDLINE
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