The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development
| Autor: | Aleksandra Shcherbakova, Karsten Cirksena, Joachim Wittbrodt, Falk F. R. Buettner, Roman Sakson, Lars R. Jensen, Thomas Ruppert, Daniel Todt, Andreas W. Kuss, Alina Friedrich, Stefan Weiss, Hermann J. Hütte, Hans Bakker, Thomas Thumberger |
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| Rok vydání: | 2021 |
| Předmět: |
RT
retention time TSR thrombospondin type 1 repeat Oryzias LTQ Linear Trap Quadrupole CBiPSC2 human cord blood–derived induced pluripotent stem cell clone 2 Eye Biochemistry Mannosyltransferases Analytical Chemistry ADAMTS Proteins hiPSC human induced pluripotent stem cell Zebrafish secretomics FA formic acid Gene Editing 0303 health sciences Metalloproteinase ESI electrospray ionization 030302 biochemistry & molecular biology ADAMTS16 RI ROCK inhibitor Cell biology Mannosylation Gene Knockdown Techniques coloboma THBS1 thrombospondin 1 sgRNA single-guide RNA Induced Pluripotent Stem Cells Biology CHO Chinese hamster ovary Cell Line TFA trifluoroacetic acid 03 medical and health sciences HEK human embryonic kidney Cricetulus C-mannosylation Thrombospondin 1 Animals Humans Secretion eGFP enhanced GFP Molecular Biology 030304 developmental biology Thrombospondin Research HEK 293 cells LFQ label-free quantification ACN acetonitrile biology.organism_classification EIC extracted ion chromatogram carbohydrates (lipids) human induced pluripotent stem cells Eye development FCS fetal calf serum Mannose MRM multiple reaction monitoring |
| Zdroj: | Molecular & Cellular Proteomics : MCP |
| ISSN: | 1535-9484 |
| Popis: | C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR–Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent stem cells using MS-based quantitative proteomics. The secretion of numerous proteins was reduced in these mutants, including that of A Disintegrin And Metalloproteinase with ThromboSpondin Motifs 16 (ADAMTS16), an extracellular protease that was previously reported to be essential for optic fissure fusion in zebrafish eye development. To test the functional relevance of this observation, we targeted dpy19l1 or dpy19l3 in embryos of the Japanese rice fish medaka (Oryzias latipes) by CRISPR–Cas9. We observed that targeting of dpy19l3 partially caused defects in optic fissure fusion, called coloboma. We further showed in a cellular model that DPY19L1 and DPY19L3 mediate C-mannosylation of a recombinantly expressed thrombospondin type 1 repeat of ADAMTS16 and thereby support its secretion. Taken together, our findings imply that DPY19L3-mediated C-mannosylation is involved in eye development by assisting secretion of the extracellular protease ADAMTS16. Graphical Abstract Highlights • TSR1 of ADAMTS16 can be C-mannosylated. • Deletion of DPY19L1 or DPY19L3 in hiPSCs caused reduced secretion of ADAMTS16. • Targeting of dpy19l3 in medaka occasionally led to coloboma. In Brief We identified ADAMTS16 as a target protein for C-mannosylation and showed that this modification is needed for proper secretion of ADAMTS16. Targeting a distinct C-mannosyltransferase by CRISPR–Cas9 in medaka fish embryos caused defects in eye development. We conclude that these developmental defects are caused by reduced secretion of ADAMTS16 when C-mannosylation is missing. |
| Databáze: | OpenAIRE |
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