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Discovery of an O-Mannosylation Pathway Selectively Serving Cadherins and Protocadherins

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Discovery of an O-Mannosylation Pathway Selectively Serving Cadherins and Protocadherins Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins Ida Signe Bohse Larsena,1, Yoshiki Narimatsua,1, Hiren Jitendra Joshia,1, Lina Siukstaitea, Oliver J. Harrisonb, Julia Braschb, Kerry M. Goodmanb, Lars Hansena, Lawrence Shapirob,c,d, Barry Honigb,c,d,e, Sergey Y. Vakhrusheva, Henrik Clausena, and Adnan Halima,2,3 aDepartment of Cellular and Molecular Medicine, Faculty of Health Sciences, Copenhagen Center for Glycomics, University of Copenhagen, DK-2200 Copenhagen, Denmark; bDepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032; cZuckerman Mind Brain Behavior Institute, Columbia University, New York, NY 10032; dDepartment of Systems Biology, Columbia University, New York, NY 10032; and eHoward Hughes Medical Institute, Columbia University, New York, NY 10032 Edited by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, and approved September 6, 2017 (received for review May 22, 2017) The cadherin (cdh) superfamily of adhesion molecules carry muscular dystrophies that have been designated α-dystroglyca- O-linked mannose (O-Man) glycans at highly conserved sites nopathies because deficient O-Man glycosylation of α-DG dis- localized to specific β-strands of their extracellular cdh (EC) domains. rupts the interaction between the dystrophin glycoprotein complex These O-Man glycans do not appear to be elongated like O-Man and the ECM (7–9). Several studies have also implicated de- glycans found on α-dystroglycan (α-DG), and we recently demon- ficiency of POMT2 with E-cdh dysfunction (10–12), although di- strated that initiation of cdh/protocadherin (pcdh) O-Man glycosyl- rect evidence for a role in glycosylation of cdhs and pcdhs is ation is not dependent on the evolutionary conserved POMT1/ missing. To explore the functions of O-Man glycans on cdhs and POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, pcdhs, we previously used a combinatorial gene-targeting strategy we used a CRISPR/Cas9 genetic dissection strategy combined with in multiple cell lines and found that the two POMTs are essential sensitive and quantitative O-Man glycoproteomics to identify a ho- for glycosylation of α-DG but not cdhs, pcdhs, and IPT/TIG domain- mologous family of four putative protein O-mannosyltransferases containing proteins (13). In contrast to α-DG, we and others (6, 10, encoded by the TMTC1–4 genes, which were found to be imperative 11, 13) also found that O-Man glycans on the latter proteins were for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in not elongated, suggesting that the biosynthesis of these distinct HEK293 cells resulted in specific loss of cdh and pcdh O-Man glyco- classes of proteins were different. We therefore predicted the ex- sylation, whereas combined KO of TMTC1 and TMTC3 resulted in istence of a new type of O-Man glycosylation machinery in higher selective loss of O-Man glycans on specific β-strands of EC domains, eukaryotes (13). suggesting that each isoenzyme serves a different function. In Here, we report the discovery of a homologous family of pu- addition, O-Man glycosylation of IPT/TIG domains of plexins and tative O-Man glycosyltransferases (GTs) encoded by the four hepatocyte growth factor receptor was not affected in TMTC transmembrane and tetra-trico-peptide repeat (TPR) repeat- KO cells, suggesting the existence of yet another O-Man glyco- containing protein (TMTC) genes (TMTC1–4). The TMTC sylation machinery. Our study demonstrates that regulation of proteins were previously shown to be located in the endoplasmic + O-mannosylation in higher eukaryotes is more complex than reticulum (ER) and predicted to serve in Ca2 regulation and envisioned, and the discovery of the functions of TMTCs provide protein folding (14, 15). Moreover, biallelic mutations in TMTC3 insight into cobblestone lissencephaly caused by deficiency were reported to cause cobblestone lissencephaly (16), intellec- in TMTC3. tual disabilities, and epilepsy (17), and family linkage and asso- ciation analysis points to a role of TMTC2 in hearing loss (18). In glycoproteomics | O-glycosylation | glycosyltransferase | mass murine models, tmtc3-KO results in early neonatal death, and spectrometry | gene editing Significance rotein O-mannosylation on select Ser/Thr residues is found Pin yeast and metazoans, and the genetic and biosynthetic The large superfamily of cadherins serve essential roles basis for this type of glycosylation has long been thought to be an in cell–cell interactions and guidance. The extracellular cad- evolutionarily conserved family of protein O-mannosyltransferases herin (EC) domains responsible for the biological functions are with seven members in yeast (Pmt1-7) and two in higher eukaryotes decorated with O-linked mannose glycans, but the functions (POMT1/POMT2) (1). O-linked mannose (O-Man) glycosylation is of these O-glycans are poorly understood. Here we describe an the only type of O-glycosylation found in yeast (2, 3), and O-Man O-mannosylation pathway orchestrated by four homologous glycans are widely found on proteins trafficking the secretory path- TMTC1–4 genes that is dedicated selectively to the cadherin way, similar to O-GalNAc glycosylation in metazoans (4). In contrast, superfamily. Mutations in the TMTC3 gene cause cobblestone O-Man glycosylation in metazoans is found only on select proteins, lissencephaly, demonstrating the importance of this type of with α-dystroglycan (α-DG) being the best characterized (5–7), O-mannosylation. but, with the advent of sensitive glycoproteomics strategies, we recently found O-Man glycans on the large superfamily of cad- Author contributions: I.S.B.L., Y.N., H.J.J., H.C., and A.H. designed research; I.S.B.L., Y.N., herins (cdhs) and protocadherins (pcdhs) as well as a family of H.J.J., L. Siukstaite, and S.Y.V. performed research; L. Shapiro and B.H. contributed new reagents/analytic tools; I.S.B.L., Y.N., H.J.J., L. Siukstaite, O.J.H., J.B., K.G., L.H., L. Shapiro, CELL BIOLOGY IPT/TIG domain-carrying proteins, including the hepatocyte B.H., S.Y.V., H.C., and A.H. analyzed data; and I.S.B.L., H.C., and A.H. wrote the paper. growth factor receptor (HGFR) (6). The O-Man glycans on cdhs The authors declare no conflict of interest. β and pcdhs are found in highly conserved positions in the -strands This article is a PNAS Direct Submission. of extracellular cdh (EC) domains, and these glycosites appear 1I.S.B.L., Y.N., and H.J.J. contributed equally to this work. highly conserved in evolution, suggesting that they serve impor- 2Present address: Laboratory of Cellular and Structural Biology, The Rockefeller Univer- tant biological functions (6). sity, New York, NY 10065. Deficiencies in enzymes catalyzing the structurally complex 3To whom correspondence should be addressed. Email: [email protected]. α and diverse O-Man glycans on -DG, including the two human This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. POMT1 and POMT2 genes, underlie a subgroup of congenital 1073/pnas.1708319114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1708319114 PNAS | October 17, 2017 | vol. 114 | no. 42 | 11163–11168 Downloaded by guest on September 28, 2021 in vitro studies have demonstrated abnormal TGF-β signaling in glycans suitable for lectin weak affinity chromatography (LWAC) embryonic fibroblasts from these mice (19). enrichment of O-Man glycopeptides with the ConA lectin as well We used combinatorial gene KO targeting in HEK293 cells as O-GalNAc with the VVA lectin (6, 13, 26) (Fig. 1A). α-DG combined with differential O-Man glycoproteomics to discover contains a central mucin-like domain with O-Man glycans in the the roles of the TMTC genes in O-Man glycosylation of cdhs N-terminal region and O-GalNAc glycans in the C-terminal re- and pcdhs, and we validated this by analysis of recombinant- gion, and is therefore a useful probe for detection of substitution expressed secreted cdh construct in TMTC mutant cell lines. We present strong evidence that the TMTC genes encode dis- tinct O-Man GTs that cooperatively glycosylate distinct regions in the EC domains of cdhs and pcdhs. We also present evidence that the TMTCs do not glycosylate the IPT/TIG domain-containing A Genetic Deconstruction Quantitative Glycoproteomics proteins, and therefore we predict the existence of yet another Dimethyl KO independent undiscovered O-Man glycosylation pathway dedicated to IPT/TIG KO labeling COSMC SC domains. The severe phenotype associated with deficiency of Mix POMGNT1 POMT1/2 TMTC3 (16, 17) indicates that O-Man glycans on the cdh super- KO KO/SC 1:1 family serve important biological functions. TMTC1,3 KO dependent HEK293WT HEK293SC KO ConA LWAC Results Gal GlcNAc TMTC1,2,3,4 KO LC-MS/MS Bioinformatic Identification of the TMTC1–4 Genes. Our finding that Man GalNAc NeuAc the POMT1/2 genes were dispensable for O-mannosylation of many proteins (13) prompted a search for additional enzymes. B Cdhs CDH11 We first tested the possibility that candidate enzyme(s) re- CDH13 sponsible for cdh/pcdh O-mannosylation were already classified as GTs in the Carbohydrate-Active enZYmes (CAZy) database, albeit Pcdhs without known function, and KO of six such genes in combinations IPT/TIG predicted to cover potential redundant isoenzymes in individual GT families did not affect O-mannosylation (GLT1D1/GTDC1, POMT1/2 GLT8D1/GLT8D2, and KDELC1/KDELC2; not shown). We substrates next hypothesized that the candidate enzyme(s) would resem- ble existing protein mannosyltransferases in CAZy GT39 and Others GT98 families with respect to overall multitransmembrane do- 0.1 1 10 100 1000 10000 100000 main structure but without significant sequence similarity. We Fold change (O-Man glycosylation) performed a broad search considering criteria common for Pmts/ C POMTs and the DPY19L genes responsible for C-mannosylation, including ER localization, general topology, and domain structure Cdhs features.
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