Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae

Neubert, Patrick; Halim, Adnan; Zauser, Martin; Essig, Andreas; Joshi, Hiren J.; Zatorska, Ewa; Larsen, Ida Signe Bohse; Loibl, Martin; Castells-Ballester, Joan; Aebi, Markus; Clausen, Henrik; Strahl, Sabine

doi:10.1074/mcp.m115.057505

Cited by 63 publications

(71 citation statements)

References 63 publications

(108 reference statements)

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“…The nucleocytoplasmic O-mannosylation system is only found in yeast and is predicted to serve similar functions as the nucleocytoplasmic O-GlcNAcylation found in all eukaryotic cells except yeast (22). The ER-located PMTs in yeast have wide glycosylation functions of ER/Golgi, cell wall, and secreted proteins, similar to the metazoan GalNAc-type O-glycosylation (23), and in fact it appears that the two types of glycosylation have great overlaps in proteins and glycosites as well as biological functions (20,24). Interestingly, the orthologous metazoans POMT1 and POMT2 are predicted to have narrower substrate specificities and only serve in glycosylating a limited number of proteins, including ␣-DG and cdhs/pcdhs (13,15,19).…”

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“…This categorization is based on sequence similarities, but a recent study confirmed the func-tional similarity between the human POMT1 and yeast PMT4 enzymes (19). O-Mannosylation of proteins in yeast is widespread, and we recently characterized the yeast O-Man glycoproteome identifying almost 300 glycoproteins that enter the secretory pathway (20). In addition, we also found that yeast has an additional and unique nucleocytoplasmic O-Man glycoproteome, which is predicted to be glycosylated by a yet unknown cytosolic/nuclear O-mannosyltransferase(s) different from the ER-located PMTs (21).…”

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Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Larsen

Narimatsu

Joshi

et al. 2017

Journal of Biological Chemistry

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Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated ␣-dystroglycanopathies, because deficient O-Man glycosylation of ␣-dystroglycan disrupts laminin interaction with ␣-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose ␤-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to ␣-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to ␣-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins.Protein O-glycosylation of the O-mannose type was originally thought to be found only in yeast and fungi, but studies over the last 30 years have identified O-Man 2 glycans and specific glycoproteins carrying O-Man glycans in human and rodents (1-9). The basement membrane glycoprotein ␣-dystroglycan (␣-DG) was for some time the only well characterized O-mannosylated protein known in mammals despite evidence that O-Man glycans constitute a major part of the total O-glycans in the brain (1,2,8,9). O-Mannosylation of ␣-DG is essential for assembly and function of the dystrophin-glycoprotein complex that links the cytoskeleton with the extracellular matrix, and deficiencies in all of the enzymes involved in the O-Man glycosylation underlie a subgroup of congenital muscular dystrophies (10 -12). More recently, the human O-Man glycoproteome was characterized, and it was found that the large superfamily of cadherins (cdhs) and protocadherins (pcdhs) are also decorated with ␣-linked O-Man glycans on extracellular cadherin (EC) domains. The attachment sites of these O-Man glycans appear to be highly conserved throughout evolution (13,14); moreover, O-mannosylation of E-cadherin was suggested to be crucial for E-cadherin-mediated cell adhesion (15,16).O-Man glycosylation in metazoans is initiated in the endoplasmic reticulum (ER) by transfer of mannose from dolichol monophosphate-activated mannose to serine and threonine by the POMT1 and POMT2 p...

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Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Larsen

Narimatsu

Joshi

et al. 2017

Journal of Biological Chemistry

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“…Interestingly, Ykl077w‐N′ runs at a much higher molecular weight than would be predicted by its length, suggesting that it is highly glycosylated (Figure B). In support of this, many sites on Ykl077w‐N′ were recently shown to be O ‐mannosylated in a high throughput analysis of glycosylation in yeast (Figure S2). We performed pull downs of Ykl077w‐N′ and Ykl077w‐C′, which demonstrated physical interactions with most of the Golgi glycosylation machinery (Tables S5 and S6).…”

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confidence: 81%

Two novel effectors of trafficking and maturation of the yeast plasma membrane H⁺‐ATPase

Geva

Crissman

Arakel

et al. 2017

Traffic

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The endoplasmic reticulum (ER) is the entry site of proteins into the endomembrane system.Proteins exit the ER via coat protein II (COPII) vesicles in a selective manner, mediated either by direct interaction with the COPII coat or aided by cargo receptors. Despite the fundamental role of such receptors in protein sorting, only a few have been identified. To further define the machinery that packages secretory cargo and targets proteins from the ER to Golgi membranes, we used multiple systematic approaches, which revealed 2 uncharacterized proteins that mediate the trafficking and maturation of Pma1, the essential yeast plasma membrane proton ATPase. Ydl121c (Exp1) is an ER protein that binds Pma1, is packaged into COPII vesicles, and whose deletion causes ER retention of Pma1. Ykl077w (Psg1) physically interacts with Exp1 and can be found in the Golgi and coat protein I (COPI) vesicles but does not directly bind Pma1. Loss of Psg1 causes enhanced degradation of Pma1 in the vacuole. Our findings suggest that Exp1 is a Pma1 cargo receptor and that Psg1 aids Pma1 maturation in the Golgi or affects its retrieval. More generally our work shows the utility of high content screens in the identification of novel trafficking components.The endoplasmic reticulum (ER) is the entry site for proteins into the endomembrane system. Once appropriately folded, non-ER resident proteins are routed to the Golgi apparatus for further maturation and distribution.1 Proteins exit the ER via coat protein II (COPII) vesicles.Although some proteins are captured by stochastic sampling of the ER lumen and membrane in a process called bulk flow, 2,3 more efficient and regulated sorting relies on selective uptake into vesicles.Additional sorting specificity occurs by retrieval of ER residents or immature proteins from the Golgi apparatus to the ER through coat protein I (COPI) vesicles. 4,5 The general mechanisms of vesicle formation are well characterized, yet the molecular basis that governs specific recognition between the coats, and the diverse range of proteins that must be packaged into vesicles, is yet to be fully described. To perturb traffic we chose to use mutations in the cargo binding sites of Sec24 as it was already shown that a mutation in the Sec24-A binding site perturbs ER-Golgi vesicular traffic. 26 Moreover, the 2 -best-characterized cargo of the A-and C-sites are SNARE proteins that act in anterograde (Sed5) and retrograde (Sec22) traffic, suggesting that bidirectional traffic between the ER and Golgi would be perturbed in these mutants. In contrast, many cargo that engage the Bsite are proteins that move forward in the secretory pathway. Thus we focused on A-and C-site mutations that should more specifically perturb ER-Golgi vesicular traffic. 6,7 We used automated mating techniques to integrate these Sec24 binding site mutants into the secretome-GFP library 27 and imaged the resulting strains using a high content setup ( Figure 1A).

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“…The mannosylated peptide identified in mouse Pdia3, includes a conserved ProThr-motif that has been demonstrated to carry an O-linked mannose in PDIA3 from human breast cancer cells [31] (S10 Fig). Most interestingly, we very recently found O-mannosyl glycans also in the ER-luminal PDIs and PDI-like proteins from baker´s yeast and found that modification by O-mannosylation occurred in a conserved residue in the proximity of the catalytic CysXXCys motifs [57]. …”

Section: Discussionmentioning

confidence: 99%

Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates

Bartels

Winterhalter

et al. 2016

PLoS ONE

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Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.

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Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae

Cited by 63 publications

References 63 publications

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Two novel effectors of trafficking and maturation of the yeast plasma membrane H⁺‐ATPase

Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates

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Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae

Cited by 63 publications

References 63 publications

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Two novel effectors of trafficking and maturation of the yeast plasma membrane H+‐ATPase

Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates

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Two novel effectors of trafficking and maturation of the yeast plasma membrane H⁺‐ATPase