Sabine Strahl

Protein O-mannosylation: Conserved from bacteria to humans*

Protein O-mannosylation is an essential modification in fungi and animals. Different from most other types of O-glycosylation, protein O-mannosylation is initiated in the endoplasmic reticulum by the transfer of mannose from dolichol monophosphate-activated mannose to serine and threonine residues of secretory proteins. In recent years, it has emerged that even bacteria are capable of O-mannosylation and that the biosynthetic pathway of O-mannosyl glycans is conserved between pro- and eukaryotes. In this review, we summarize the observations that have opened up the field and highlight characteristics of O-mannosylation in the different domains/kingdoms of life.

ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker-Warburg syndrome

Walker-Warburg syndrome (WWS) is clinically defined as congenital muscular dystrophy that is accompanied by a variety of brain and eye malformations. It represents the most severe clinical phenotype in a spectrum of diseases associated with abnormal post-translational processing of α-dystroglycan that share a defect in laminin-binding glycan synthesis. Although mutations in six genes have been identified as causes of WWS, only half of all individuals with the disease can currently be diagnosed on this basis. A cell fusion complementation assay in fibroblasts from undiagnosed individuals with WWS was used to identify five new complementation groups. Further evaluation of one group by linkage analysis and targeted sequencing identified recessive mutations in the ISPD gene (encoding isoprenoid synthase domain containing). The pathogenicity of the identified ISPD mutations was shown by complementation of fibroblasts with wild-type ISPD. Finally, we show that recessive mutations in ISPD abolish the initial step in laminin-binding glycan synthesis by disrupting dystroglycan O-mannosylation. This establishes a new mechanism for WWS pathophysiology.

Members of the evolutionarily conserved PMT family of protein O-mannosyltransferases form distinct protein complexes among themselves.

Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification. Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi. The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal. In this study we analyzed the molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance ofO-mannosylation. Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex. Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general. Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall proteinO-mannosylation in fungi.

Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants.

ABSTRACT Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Δ mutant. We found that pmt2 pmt4Δ cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Δ mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Δ mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.

Protein O-mannosylation is crucial for cell wall integrity, septation and viability in fission yeast.

Protein O‐mannosyltransferases (PMTs) initiate the assembly of O‐mannosyl glycans, which are of fundamental importance in eukaryotes. The PMT family, which is classified into PMT1, PMT2 and PMT4 subfamilies, is evolutionarily conserved. Despite the fact that PMTs are crucial for viability of bakers yeast as well as of mouse, recent studies suggested that there are significant differences in the organization and properties of the O‐mannosylation machinery between yeasts and mammals. In this study we identified and characterized the PMT family of the archaeascomycete Schizosaccharomyces pombe. Unlike Saccharomyces cerevisiae where the PMT family is highly redundant, in S. pombe only one member of each PMT subfamily is present, namely, oma1+ (protein O‐mannosyltransferase), oma2+ and oma4+. They all act as protein O‐mannosyltransferases in vivo. oma1+ and oma2+ form heteromeric protein complexes and recognize different protein substrates compared to oma4+, suggesting that similar principles underlie mannosyltransfer reaction in S. pombe and budding yeast. Deletion of oma2+, as well as simultaneous deletion of oma1+ and oma4+ is lethal. Characterization of the viable S. pombe oma1Δ and oma4Δ single mutants showed that a lack of O‐mannosylation results in abnormal cell wall and septum formation, thereby severely affecting cell morphology and cell–cell separation.

O‐Mannosylation precedes and potentially controls the N‐glycosylation of a yeast cell wall glycoprotein

Secretory proteins in yeast are N‐ and O‐glycosylated while they enter the endoplasmic reticulum. N‐glycosylation is initiated by the oligosaccharyl transferase complex and O‐mannosylation is initiated by distinct O‐mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell‐wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O‐mannosylation by Pmt4 prevents N‐glycosylation by blocking the hydroxy amino acid of the single N‐glycosylation site present in Ccw5. These data prove that the O‐ and N‐glycosylation machineries compete for Ccw5; therefore O‐mannosylation by Pmt4 precedes N‐glycosylation.

Members of protein O‐mannosyltransferase family in Aspergillus fumigatus differentially affect growth, morphogenesis and viability

O‐mannosylation is an essential protein modification in eukaryotes. It is initiated at the endoplasmic reticulum by O‐mannosyltransferases (PMT) that are evolutionary conserved from yeast to humans. The PMT family is phylogenetically classified into PMT1, PMT2 and PMT4 subfamilies, which differ in protein substrate specificity and number of genes per subfamily. In this study, we characterized for the first time the whole PMT family of a pathogenic filamentous fungus, Aspergillus fumigatus. Genome analysis showed that only one member of each subfamily is present in A. fumigatus, PMT1, PMT2 and PMT4. Despite the fact that all PMTs are transmembrane proteins with conserved peptide motifs, the phenotype of each PMT deletion mutant was very different in A. fumigatus. If disruption of PMT1 did not reveal any phenotype, deletion of PMT2 was lethal. Disruption of PMT4 resulted in abnormal mycelial growth and highly reduced conidiation associated to significant proteomic changes. The double pmt1pmt4 mutant was lethal. The single pmt4 mutant exhibited an exquisite sensitivity to echinocandins that is associated to major changes in the expression of signal transduction cascade genes. These results indicate that the PMT family members play a major role in growth, morphogenesis and viability of A. fumigatus.

Protein O-mannosylation: What we have learned from baker's yeast

BACKGROUND Protein O-mannosylation is a vital type of glycosylation that is conserved among fungi, animals, and humans. It is initiated in the endoplasmic reticulum (ER) where the synthesis of the mannosyl donor substrate and the mannosyltransfer to proteins take place. O-mannosylation defects interfere with cell wall integrity and ER homeostasis in yeast, and define a pathomechanism of severe neuromuscular diseases in humans. SCOPE OF REVIEW On the molecular level, the O-mannosylation pathway and the function of O-mannosyl glycans have been characterized best in the eukaryotic model yeast Saccharomyces cerevisiae. In this review we summarize general features of protein O-mannosylation, including biosynthesis of the mannosyl donor, characteristics of acceptor substrates, and the protein O-mannosyltransferase machinery in the yeast ER. Further, we discuss the role of O-mannosyl glycans and address the question why protein O-mannosylation is essential for viability of yeast cells. GENERAL SIGNIFICANCE Understanding of the molecular mechanisms of protein O-mannosylation in yeast could lead to the development of novel antifungal drugs. In addition, transfer of the knowledge from yeast to mammals could help to develop diagnostic and therapeutic approaches in the frame of neuromuscular diseases. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

Protein O-mannosylation is crucial for E-cadherin-mediated cell adhesion

Significance Cell–cell adhesion is essential for embryonic development, tissue morphogenesis, and tissue repair, as well as for tumor invasion and metastasis. Thus, it is of fundamental importance to identify the molecular factors that affect this process. Here we demonstrate that O-mannosylation, an essential posttranslational protein modification, is crucial for the formation of adherens junctions between cells of the early mouse embryo, with O-mannosylation–deficient embryos dying at the morula-to-blastocyst transition. Moreover, we identified O-mannosyl glycans on E-cadherin, the major cell–cell adhesion protein of embryos, and demonstrated that these glycans are crucial for E-cadherin–mediated cell adhesion. Because O-mannosylation is a conserved feature of the classical cadherins, this protein modification most likely affects many more biological processes than previously thought. In recent years protein O-mannosylation has become a focus of attention as a pathomechanism underlying severe congenital muscular dystrophies associated with neuronal migration defects. A key feature of these disorders is the lack of O-mannosyl glycans on α-dystroglycan, resulting in abnormal basement membrane formation. Additional functions of O-mannosylation are still largely unknown. Here, we identify the essential cell–cell adhesion glycoprotein epithelial (E)-cadherin as an O-mannosylated protein and establish a functional link between O-mannosyl glycans and cadherin-mediated cell–cell adhesion. By genetically and pharmacologically blocking protein O-mannosyltransferases, we found that this posttranslational modification is essential for preimplantation development of the mouse embryo. O-mannosylation–deficient embryos failed to proceed from the morula to the blastocyst stage because of defects in the molecular architecture of cell–cell contact sites, including the adherens and tight junctions. Using mass spectrometry, we demonstrate that O-mannosyl glycans are present on E-cadherin, the major cell-adhesion molecule of blastomeres, and present evidence that this modification is generally conserved in cadherins. Further, the use of newly raised antibodies specific for an O-mannosyl–conjugated epitope revealed that these glycans are present on early mouse embryos. Finally, our cell-aggregation assays demonstrated that O-mannosyl glycans are crucial for cadherin-based cell adhesion. Our results redefine the significance of O-mannosylation in humans and other mammals, showing the immense impact of cadherins on normal as well as pathogenic cell behavior.

Membrane association is a determinant for substrate recognition by PMT4 protein O-mannosyltransferases

Protein O-mannosylation represents an evolutionarily conserved, essential posttranslational modification with immense impact on a variety of cellular processes. In humans, O-mannosylation defects result in Walker–Warburg syndrome, a severe recessive congenital muscular dystrophy associated with defects in neuronal migration that produce complex brain and eye abnormalities. In mouse and yeasts, loss of O-mannosylation causes lethality. Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans. The evolutionarily conserved PMT family is classified into PMT1, PMT2, and PMT4 subfamilies, which mannosylate distinct target proteins. In contrast to other types of glycosylation, signal sequences for O-mannosylation have not been identified to date. In the present study, we identified signals that determine PMT4-dependent O-mannosylation. Using specific model proteins, we demonstrate that in yeast Pmt4p mediates O-mannosylation of Ser/Thr-rich membrane-attached proteins. The nature of the membrane-anchoring sequence is nonrelevant, as long as it is flanked by a Ser/Thr-rich domain facing the endoplasmic reticulum lumen. Our work shows that, in contrast to several other types of glycosylation, PMT4 O-mannosylation signals are not just linear proteins primary structure sequences but rather are highly complex. Based on these findings, we performed in silico analyses of the Saccharomyces cerevisiae proteome and identified previously undescribed Pmt4p substrates. This tool for proteome-wide identification of O-mannosylated proteins is of general interest because several of these proteins are major players of a wide variety of cellular processes.