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Glycobiology

Prof. Dr. Sabine Strahl

2016:

Bartels MF, Winterhalter PR, Yu J, Liu Y, Lommel M, Möhrlen F, Hu H, Feizi T, Westerlind U, Ruppert T, Strahl S. (2016). Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates. PLoS One. 11(11):e0166119.

Abstract
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.
 

 

Bausewein D, Engel J, Jank T, Schoedl M, Strahl S. (2016). Functional Similarities between the Protein O-Mannosyltransferases Pmt4 from Bakers' Yeast and Human POMT1. J Biol Chem. 291(34):18006-15.

Abstract
Protein O-mannosylation is an essential post-translational modification. It is initiated in the endoplasmic reticulum by a family of protein O-mannosyltransferases that are conserved from yeast (PMTs) to human (POMTs). The degree of functional conservation between yeast and human protein O-mannosyltransferases is uncharacterized. In bakers' yeast, the main in vivo activities are due to heteromeric Pmt1-Pmt2 and homomeric Pmt4 complexes. Here we describe an enzymatic assay that allowed us to monitor Pmt4 activity in vitro We demonstrate that detergent requirements and acceptor substrates of yeast Pmt4 are different from Pmt1-Pmt2, but resemble that of human POMTs. Furthermore, we mimicked two POMT1 amino acid exchanges (G76R and V428D) that result in severe congenital muscular dystrophies in humans, in yeast Pmt4 (I112R and I435D). In vivo and in vitro analyses showed that general features such as protein stability of the Pmt4 variants were not significantly affected, however, the mutants proved largely enzymatically inactive. Our results demonstrate functional and biochemical similarities between POMT1 and its orthologue from bakers' yeast Pmt4.

Pubmed 

 

Carvalho S, Oliveira T, Bartels MF, Miyoshi E, Pierce M, Taniguchi N, Carneiro F, Seruca R, Reis CA, Strahl S, Pinho SS (2016) O-mannosylation and N-glycosylation: two coordinated mechanisms regulating the tumour suppressor functions of E-cadherin in cancer.  Oncotarget. [Epub ahead of print]

Abstract
Dysregulation of tumor suppressor protein E-cadherin is an early molecular event in cancer. O-mannosylation profile of E-cadherin is a newly-described post-translational modification crucial for its adhesive functions in homeostasis. However, the role of O-mannosyl glycans in E-cadherin-mediated cell adhesion in cancer and their interplay with N-glycans remains largely unknown. We herein demonstrated that human gastric carcinomas exhibiting a non-functional E-cadherin display a reduced expression of O-mannosyl glycans concomitantly with increased modification with branched complex N-glycans. Accordingly, overexpression of MGAT5-mediated branched N-glycans both in gastric cancer cells and transgenic mice models led to a significant decrease of O-mannosyl glycans attached to E-cadherin that was associated with impairment of its tumour suppressive functions. Importantly, overexpression of protein O-mannosyltransferase 2 (POMT2) induced a reduced expression of branched N-glycans which led to a protective effect of E-cadherin biological functions. Overall, our results reveal a newly identified mechanism of (dys)regulation of E-cadherin that occur through the interplay between O-mannosylation and N-glycosylation pathway.

Pubmed 

 

Neubert P and Strahl S. (2016) Protein O-mannosylation in the early secretory pathway. Curr Opin Cell Biol 41:100-8. doi: 10.1016/j.ceb.2016.04.010. Review.

Abstract
Protein O-mannosylation and N-glycosylation are essential post-translational modifications, which initiate in the endoplasmic reticulum (ER). In yeast, the two glycosylation machineries act at the Sec61 translocon complex where they can even compete for certain substrate proteins. N-linked glycans play a crucial role in the ER quality control of glycoproteins. In recent years, it became clear that in addition to its important functions for cell surface proteins, O-mannosylation impacts the ER protein homeostasis. These glycans can exclude unfavorable folding intermediates from futile folding attempts, increase the solubility of irreversibly misfolded proteins, and even mark them for degradation. O-Mannose glycoproteomics now captures the molecular complexity of this modification opening exciting opportunities to explore further roles of O-mannosylation in the early secretory pathway.

Pubmed 

 

Neubert, P, Halim, A, Zauser, M, Essig, A, Joshi, HJ, Zatorska, E, Larsen, IS, Loibl, M, Castells-Ballester, J, Aebi, M, Clausen, H and Strahl, S (2016). Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae. Mol Cell Proteomics. 15,1323-37.

Abstract
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.

Pubmed 

 

2015:

Ragni E, Lommel M, Moro M, Crosti M, Lavazza C, Parazzi V, Saredi S, Strahl S, Lazzari L. (2015). Protein O-mannosylation is crucial for human mesencyhmal stem cells fate. Cell Mol Life Sci. 73(2):445-58.

Abstract
Human mesenchymal stem cells (MSC) are promising cell types in the field of regenerative medicine. Although many pathways have been dissected in the effort to better understand and characterize MSC potential, the impact of protein N- or O-glycosylation has been neglected. Deficient protein O-mannosylation is a pathomechanism underlying severe congenital muscular dystrophies (CMD) that start to develop at the embryonic developmental stage and progress in the adult, often in tissues where MSC exert their function. Here we show that O-mannosylation genes, many of which are putative or verified glycosyltransferases (GTs), are expressed in a similar pattern in MSC from adipose tissue, bone marrow, and umbilical cord blood and that their expression levels are retained constant during mesengenic differentiation. Inhibition of the first players of the enzymatic cascade, POMT1/2, resulted in complete abolishment of chondrogenesis and alterations of adipogenic and osteogenic potential together with a lethal effect during myogenic induction. Since to date, no therapy for CMD is available, we explored the possibility of using MSC extracellular vesicles (EVs) as molecular source of functional GTs mRNA. All MSC secrete POMT1 mRNA-containing EVs that are able to efficiently fuse with myoblasts which are among the most affected cells by CMD. Intriguingly, in a pomt1 patient myoblast line EVs were able to partially revert O-mannosylation deficiency and contribute to a morphology recovery. Altogether, these results emphasize the crucial role of protein O-mannosylation in stem cell fate and properties and open the possibility of using MSC vesicles as a novel therapeutic approach to CMD.

Pubmed 

 

Halim A, Larsen IS, Neubert P, Joshi HJ, Petersen BL, Vakhrushev SY, Strahl S, Clausen H.  (2015)  Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast. Proc Natl Acad Sci U S A112:15648-53.

Abstract
Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing.

Pubmed 

 

2014:

Winterhalter PR, Ruppert T, Strahl S. (2014)  Identification of Mammalian O-Mannosylated Glycopeptides. N. Taniguch et al. (eds) Glycoscience: Biology and Medicine, DOI 10.1007/978-4-431-54836-2_66-1

Abstract
O-Mannosylation is a vital protein modification that has crucial functions in cell-cell as well as cell-extracellular matrix adhesion in mammals. Although O-mannosyl glycans are abundant in the mammalian brain, the characterization of proteins bearing these glycans has been challenging. In this chapter, a straightforward LC-MS-based method involving α-mannosidase treatment is described to conclusively identify O-mannosylated proteins isolated from mammalian tissues.

Springer 

 

Loibl M, Wunderle L, Hutzler J, Schulz BL, Aebi M, Strahl S. (2014). Protein O-mannosyltransferases associate with the translocon to modify translocating polypeptide chains. J Biol Chem. 289(12):8599-611.

Abstract
O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.

Pubmed 

 

2013:

Lommel M, Winterhalter PR, Willer T, Dahlhoff M, Schneider MR, Bartels MF, Renner-Müller I, Ruppert T, Wolf E, Strahl S. (2013). Protein O-mannosylation is crucial for E-cadherin-mediated cell adhesion. Proc Natl Acad Sci U S A. 110(52):21024-9.
Abstract
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.
Pubmed 
Winterhalter PR, Lommel M, Ruppert T, Strahl S. (2013). O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues. FEBS Lett. 587(22):3715-21.
Abstract
O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC-MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.
Pubmed 
Loibl M, Strahl S. (2013). Protein O-mannosylation: what we have learned from baker's yeast. Biochim Biophys Acta. 1833(11):2438-46.
Abstract
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.
Pubmed 
Loibl M, Strahl S. (2013). Photoaffinity labeling of protein O-mannosyltransferases of the PMT1/PMT2 subfamily. Methods Mol Biol. 1022:107-17.
Abstract
Protein O-mannosylation is initiated at the endoplasmic reticulum (ER) by dolichyl phosphate-mannose: protein O-mannosyltransferases (PMTs). PMTs are members of the glycosyltransferase (GT) C superfamily. They are large polytopic integral membrane proteins located in the ER membrane. PMTs utilize dolichyl phosphate--activated mannose as sugar donor. Glycosyltransfer of mannose to serine and threonine residues of nascent polypeptides leads to an inversion of the stereochemistry of the glycosidic bond. Here, we describe photoaffinity labeling of yeast Pmt1p using a photo-reactive probe that is based on the artificial mannosyl acceptor peptide YATAV. Due to the high homology of PMTs, this method can also be applied to study PMT1 and PMT2 subfamily members from fungi other than baker's yeast.

Pubmed 

 

2012:

Willer T, Lee H, Lommel M, Yoshida-Moriguchi T, de Bernabe DB, Venzke D, Cirak S, Schachter H, Vajsar J, Voit T, Muntoni F, Loder AS, Dobyns WB, Winder TL, Strahl S, Mathews KD, Nelson SF, Moore SA, Campbell KP. (2012). ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker-Warburg syndrome. Nat Genet. 44(5):575-80.
Abstract
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 a-dystroglycan that share a defect in laminin-binding glycan synthesis1. 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 basis2. 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.

Pubmed 

 

2011:

Lommel M, Schott A, Jank T, Hofmann V, Strahl S. (2011). A conserved acidic motif is crucial for enzymatic activity of protein O-mannosyltransferases. J Biol Chem. 286(46):39768-75.
Abstract
Protein O-mannosylation is an essential modification in fungi and mammals. It is initiated at the endoplasmic reticulum by a conserved family of dolichyl phosphate mannose-dependent protein O-mannosyltransferases (PMTs). PMTs are integral membrane proteins with two hydrophilic loops (loops 1 and 5) facing the endoplasmic reticulum lumen. Formation of dimeric PMT complexes is crucial for mannosyltransferase activity, but the direct cause is not known to date. In bakers' yeast, O-mannosylation is catalyzed largely by heterodimeric Pmt1p-Pmt2p and homodimeric Pmt4p complexes. To further characterize Pmt1p-Pmt2p complexes, we developed a photoaffinity probe based on the artificial mannosyl acceptor substrate Tyr-Ala-Thr-Ala-Val. The photoreactive probe was preferentially cross-linked to Pmt1p, and deletion of the loop 1 (but not loop 5) region abolished this interaction. Analysis of Pmt1p loop 1 mutants revealed that especially Glu-78 is crucial for binding of the photoreactive probe. Glu-78 belongs to an Asp-Glu motif that is highly conserved among PMTs. We further demonstrate that single amino acid substitutions in this motif completely abolish activity of Pmt4p complexes. In contrast, both acidic residues need to be exchanged to eliminate activity of Pmt1p-Pmt2p complexes. On the basis of our data, we propose that the loop 1 regions of dimeric complexes form part of the catalytic site.
Pubmed 
Arroyo J, Hutzler J, Bermejo C, Ragni E, García-Cantalejo J, Botías P, Piberger H, Schott A, Sanz AB, Strahl S. (2011). Functional and genomic analyses of blocked protein O-mannosylation in baker's yeast. Mol Microbiol. 79(6):1529-46.
Abstract
O-mannosylation is a crucial protein modification in eukaryotes that is initiated by the essential family of protein O-mannosyltransferases (PMTs). Here we demonstrate that in the model yeast Saccharomyces cerevisiae rhodanine-3-acetic acid derivatives affect members of all PMT subfamilies. Specifically, we used OGT2468 to analyse genome-wide transcriptional changes in response to general inhibition of O-mannosylation in baker's yeast. PMT inhibition results in the activation of the cell wall integrity (CWI) pathway. Coinciding, the mitogen-activated kinase Slt2p is activated in vivo and CWI pathway mutants are hypersensitive towards OGT2468. Further, induction of many target genes of the unfolded protein response (UPR) and ER-associated protein degradation (ERAD) is observed. The interdependence of O-mannosylation and UPR/ERAD is confirmed by genetic interactions between HAC1 and PMTs, and increased degradation of the ERAD substrate Pdr5p* in pmtΔ mutants. Transcriptome analyses further suggested that mating and filamentous growth are repressed upon PMT inhibition. Accordingly, in vivo mating efficiency and invasive growth are considerably decreased upon OGT2468 treatment. Quantitative PCR and ChIP analyses suggest that downregulation of mating genes is dependent on the transcription factor Ste12p. Finally, inhibitor studies identified a role of the Ste12p-dependent vegetative signalling cascade in the adaptive response to inhibition of O-mannosylation.
Pubmed 
Ragni E, Piberger H, Neupert C, García-Cantalejo J, Popolo L, Arroyo J, Aebi M, Strahl S. (2011). The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections. BMC Genomics. 12:107.
Abstract
Mannoproteins construct the outer cover of the fungal cell wall. The covalently linked cell wall protein Ccw12p is an abundant mannoprotein. It is considered as crucial structural cell wall component since in baker's yeast the lack of CCW12 results in severe cell wall damage and reduced mating efficiency. RESULTS: In order to explore the function of CCW12, we performed a Synthetic Genetic Analysis (SGA) and identified genes that are essential in the absence of CCW12. The resulting interaction network identified 21 genes involved in cell wall integrity, chitin synthesis, cell polarity, vesicular transport and endocytosis. Among those are PFD1, WHI3, SRN2, PAC10, FEN1 and YDR417C, which have not been related to cell wall integrity before. We correlated our results with genetic interaction networks of genes involved in glucan and chitin synthesis. A core of genes essential to maintain cell integrity in response to cell wall stress was identified. In addition, we performed a large-scale transcriptional analysis and compared the transcriptional changes observed in mutant ccw12Δ with transcriptomes from studies investigating responses to constitutive or acute cell wall damage. We identified a set of genes that are highly induced in the majority of the mutants/conditions and are directly related to the cell wall integrity pathway and cell wall compensatory responses. Among those are BCK1, CHS3, EDE1, PFD1, SLT2 and SLA1 that were also identified in the SGA. In contrast, a specific feature of mutant ccw12Δ is the transcriptional repression of genes involved in mating. Physiological experiments substantiate this finding. Further, we demonstrate that Ccw12p is present at the cell periphery and highly concentrated at the presumptive budding site, around the bud, at the septum and at the tip of the mating projection. CONCLUSIONS: The combination of high throughput screenings, phenotypic analyses and localization studies provides new insight into the function of Ccw12p. A compensatory response, culminating in cell wall remodelling and transport/recycling pathways is required to buffer the loss of CCW12. Moreover, the enrichment of Ccw12p in bud, septum and mating projection is consistent with a role of Ccw12p in preserving cell wall integrity at sites of active growth.The microarray data produced in this analysis have been submitted to NCBI GEO database and GSE22649 record was assigned.
Pubmed 
Schott A, Strahl S. (2011) Methods to study stromal-cell derived factor 2 in the context of ER stress and the unfolded protein response in Arabidopsis thaliana. Methods Enzymol. 490:295-319.
Abstract
The accumulation of misfolded or unfolded polypeptides in the endoplasmic reticulum (ER) provokes ER stress and triggers protective signaling pathways termed the unfolded protein response (UPR). Stromal cell-derived factor 2 (SDF2)-type proteins are conserved throughout the animal and plant kingdoms. Upon UPR activation transcription of SDF2-type genes is significantly enhanced in metazoan and plants, suggesting an evolutionarily conserved role. However, the precise molecular function of SDF2-type proteins still needs to be established. Most eukaryotes have two SDF2 homologous, whereas the model plant Arabidopsis thaliana has a single SDF2, thus representing an ideal model system to study the functional role of SDF2-type proteins. This chapter provides techniques to study SDF2 in the context of ER stress in Arabidopsis. We describe available sdf2 mutants, and methods to evaluate ER stress sensitivity of seedlings. Further, we summarize tools and methods that are helpful to monitor UPR induction in general (e.g., SDF2 promoter-reporter fusion constructs and SDF2-specific antibodies). In Section 6, we provide protocols for the expression and purification of recombinant SDF2 protein that can be used for further biochemical studies.

Pubmed 

 

2010:

Lommel M, Willer T, Cruces J, Strahl S. (2010). POMT1 is essential for protein O-mannosylation in mammals.  Methods Enzymol. 2010;479:323-42.
Abstract
Over the past decade it has emerged that O-mannosyl glycans are not restricted to yeast and fungi but are also present in higher eukaryotes up to humans. In mammals, the protein O-mannosyltransferases POMT1 and POMT2 act as a heteromeric complex to initiate O-mannosylation in the endoplasmic reticulum. In humans, mutations in POMT1 and POMT2 result in hypoglycosylation of alpha-dystroglycan (alpha-DG) thereby abolishing its binding to extracellular matrix ligands such as laminin. As a consequence, POMT mutations cause a heterogeneous group of severe recessive congenital muscular dystrophies in humans. However, little is known about the function of O-mannosyl glycans in mammals apart from its crucial role for the ligand binding abilities of alpha-DG. In this chapter we discuss the methods used to analyze the expression of Pomt1 in adult mouse organs and during embryo development. Further, we describe the generation and immunohistochemical analysis of Pomt1 knockout mice.
Pubmed 
Schott A, Ravaud S, Keller S, Radzimanowski J, Viotti C, Hillmer S, Sinning I, Strahl S. (2010). Arabidopsis stromal-derived Factor2 (SDF2) is a crucial target of the unfolded protein response in the endoplasmic reticulum. J Biol Chem. 285(23):18113-21.
Abstract
Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.
Pubmed 
Lommel M, Cirak S, Willer T, Hermann R, Uyanik G, van Bokhoven H, Körner C, Voit T, Bari? I, Hehr U, Strahl S. (2010). Correlation of enzyme activity and clinical phenotype in POMT1-associated dystroglycanopathies. Neurology. 74(2):157-64.
Abstract
BACKGROUND: Mutations in protein O-mannosyltransferases (POMTs) cause a heterogeneous group of muscular dystrophies with abnormal glycosylation of alpha-dystroglycan (dystroglycanopathies). The wide spectrum of clinical severities ranges from Walker-Warburg syndrome (WWS), associated with brain and eye abnormalities, to mild forms of limb girdle muscular dystrophy (LGMD). OBJECTIVE: The aim of this study was to elucidate the impact of mutations in POMT1 on the clinical phenotype. METHODS: We examined 2 patients with POMT1-associated alpha-dystroglycanopathy, 1 displaying a LGMD2K and 1 with a WWS phenotype. Using dermal fibroblasts, we analyzed the influence of the POMT1 mutations on the glycosylation status of alpha-dystroglycan, protein O-mannosyltransferase activity, and the stability of the mutant POMT1 protein. RESULTS: We report on novel compound heterozygous mutations in POMT1 (p.L171A and p.A589VfsX38) that result in LGMD2K. We further demonstrate that a homozygous splice site mutation of a recently identified WWS patient results in POMT1 p.del77-93. Using dermal fibroblasts, we show that mannosyltransferase activity is reduced in the patients and that stability of POMT1 mutant proteins p.A589VfsX38 and p.del77-93 is significantly decreased. CONCLUSIONS: Our results suggest that dermal fibroblasts can be applied to facilitate the diagnostic analysis of dystroglycanopathy patients as well as to study the pathogenic mechanism of POMT mutations. Characterization of the POMT1 substrate protein alpha-dystroglycan and POMT in vitro mannosyltransferase activity shows that the severity of the clinical phenotype of the patients analyzed is inversely correlated with POMT activity.

Pubmed 

 

2009:

Radzimanowski J, Ravaud S, Schott A, Strahl S, Sinning I. (2009). Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of SDF2-like protein from Arabidopsis thaliana. Acta Crystallogr Sect F Struct Biol Cryst Commun. 66(Pt 1):12-4.
Abstract
The stromal-cell-derived factor 2-like protein of Arabidopsis thaliana (AtSDL) has been shown to be highly up-regulated in response to unfolded protein response (UPR) inducing reagents, suggesting that it plays a crucial role in the plant UPR pathway. AtSDL has been cloned, overexpressed, purified and crystallized using the vapour-diffusion method. Two crystal forms have been obtained under very similar conditions. The needle-shaped crystals did not diffract X-rays, while the other form diffracted to 1.95 A resolution using a synchrotron-radiation source and belonged to the hexagonal space group P6(1), with unit-cell parameters a = b = 96.1, c = 69.3 A.
Pubmed 
Lommel M, Strahl S. (2009). Protein O-mannosylation: conserved from bacteria to humans. Glycobiology. 19(8):816-28.
Abstract
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.

Pubmed 

 

2008:

Lommel M, Willer T, Strahl S. (2008). POMT2, a key enzyme in Walker-Warburg syndrome: somatic sPOMT2, but not testis-specific tPOMT2, is crucial for mannosyltransferase activity in vivo. Glycobiology. 18(8):615-25.
Abstract
O-Mannosylation represents an evolutionarily conserved, essential protein modification. In mammals the protein O-mannosyltransferases POMT1 and POMT2 act as a heteromeric complex to initiate O-mannosylation in the endoplasmic reticulum. Mutations in human POMT1 and POMT2 cause a group of congenital muscular dystrophies due to reduced O-glycosylation of alpha-dystroglycan. The most severe of these autosomal recessive conditions is Walker-Warburg syndrome (WWS) with severe brain and ocular involvement. We previously showed in the murine model that Pomt1 is expressed in WWS-related tissues both during embryogenesis and in adults. Whereas there is only a single Pomt1 transcript in adult mice, we demonstrated that there are two Pomt2 transcripts, somatic sPomt2 and testis-specific tPomt2. In this study we demonstrate that sPomt2, but not tPomt2, is prominently expressed in mouse embryos in the tissues that are most severely affected in WWS (developing muscle, eye, and brain). Correlation of POMT transcripts and protein isoforms with POMT mannosyltransferase enzyme activity demonstrates that sPOMT2-POMT1 complexes catalyze mannosyltransfer in adult somatic tissues and testis. It is suggested that the gonadal defects described in some WWS cases are associated with defects in O-mannosylation. Our data further show that whereas sPOMT2 is widely expressed, tPOMT2 is restricted to the acrosome of male germ cells and is not involved in the biosynthesis of O-mannosyl glycans in vivo. We prove that tPOMT2 is highly conserved among mammals, including humans, suggesting a crucial function that is distinct from sPOMT2.
Pubmed 
 
Hutzler F, Gerstl R, Lommel M, Strahl S. (2008). Protein N-glycosylation determines functionality of the Saccharomyces cerevisiae cell wall integrity sensor Mid2p. Mol Microbiol. 68(6):1438-49.
Abstract
The fungal cell wall is a highly dynamic structure that is essential to maintain cell shape and stability. Hence in yeasts and fungi cell wall integrity is tightly controlled. The Saccharomyces cerevisiae plasma membrane protein Mid2p is a putative mechanosensor that responds to cell wall stresses and morphological changes during pheromone induction. The extracellular domain of Mid2p, which is crucial to sensing, is highly O- and N-glycosylated. We showed that O-mannosylation is determining stability of Mid2p. If and how N-glycosylation is linked to Mid2p function was unknown. Here we demonstrate that Mid2p contains a single high mannose N-linked glycan at position Asn-35. The N-glycan is located close to the N-terminus and is exposed from the plasma membrane towards the cell wall through a highly O-mannosylated domain that is predicted to adopt a rod-like conformation. In contrast to O-mannosylation, lack of the N-linked glycan affects neither, stability of Mid2p nor distribution at the plasma membrane during vegetative and sexual growth. However, non-N-glycosylated Mid2p fails to perceive cell wall challenges. Our data further demonstrate that both the extent of the N-linked glycan and its distance from the plasma membrane affect Mid2p function, suggesting the N-glycan to be directly involved in Mid2p sensing.

Pubmed 

 

2007:

Hutzler J, Schmid M, Bernard T, Henrissat B, Strahl S. (2007). Membrane association is a determinant for substrate recognition by PMT4 protein O-mannosyltransferases. Proc Natl Acad Sci U S A. 104(19):7827-32.
Abstract
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 protein's 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.
Pubmed 
Ragni E, Sipiczki M, Strahl S. (2007). Characterization of Ccw12p, a major key player in cell wall stability of Saccharomyces cerevisiae. Yeast. 24(4):309-19.
Abstract
The GPI-anchored mannoprotein Ccw12p is a crucial structural component of the cell wall of Saccharomyces cerevisiae. Compared to wild-type, the mutant ccw12Δ grows more slowly, is highly sensitive to Calcofluor white and contains 2.5 times more cell wall chitin. In this study, electron microscopy of ccw12Δ cell walls revealed that, with respect to wild-type, the inner glucan layer is thicker with irregular depositions of wall material, whereas the outer mannan layer is less condensed. Biochemical analyses of cell wall glucan suggest that in the absence of Ccw12p, GPI-anchored cell wall proteins are transferred preferentially to chitin and random deposition of cell wall material reinforces the inner glucan-chitin layer, thereby enhancing the overall stability of the cell wall. To further elucidate the role of Ccw12p, structure-function analysis was performed. We demonstrate that Ccw12p is highly N-glycosylated. However, loss of N-glycans does not affect Ccw12p functionality. In contrast, deletion of the repeated amino acid motive TTEAPKNGTSTAAP in the C-terminal part of the protein affects Ccw12p function.

Pubmed 

 

2006 & older:

Lehle L, Strahl S, Tanner W. (2006). Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases. Angew Chem Int Ed Engl. 45(41):6802-18.
Abstract
Proteins can be modified by a large variety of covalently linked saccharides. The present review concentrates on two types, protein N-glycosylation and protein O-mannosylation, which, with only a few exceptions, are evolutionary conserved from yeast to man. They are also distinguished by some special features: The corresponding glycosylation processes start in the endoplasmatic reticulum, are continued in the Golgi apparatus, and require dolichol-activated precursors for the initial biosynthetic steps. With respect to the molecular biology of both types of protein glycosylation, the pathways and the genetic background of the reactions have most successfully been studied with the genetically easy-to-handle baker's yeast, Saccharomyces cerevisae. Many of the severe developmental disturbances in children are related to protein glycosylation, for example, the CDG syndrome (congenital disorders of glycosylation) as well as congenital muscular dystrophies with neuronal-cell-migration defects have been elucidated with the help of yeast.
Pubmed 
Willer T, Brandl M, Sipiczki M, Strahl S. (2005). Protein O-mannosylation is crucial for cell wall integrity, septation and viability in fission yeast. Mol Microbiol. 57(1):156-70.
Abstract
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 baker's 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.
Pubmed 
Sestak S, Hagen I, Tanner W, Strahl S. (2004). Scw10p, a cell-wall glucanase/transglucosidase important for cell-wall stability in Saccharomyces cerevisiae. Microbiology. 150(Pt 10):3197-208.
Abstract
Glycosyl hydrolases and transferases are crucial for the formation of a rigid but at the same time plastic cell wall in yeasts and fungi. The Saccharomyces cerevisiae glucan hydrolase family 17 (GH17) contains the soluble cell-wall proteins Scw4p, Scw10p, Scw11p and Bgl2p. For Bgl2p, endoglucanase/glucanosyltransferase activity has been demonstrated, and Scw11p has been shown to be involved in cell separation. Here, Scw4p and Scw10p, which show 63 % amino acid identity, were characterized. scw4 and scw10 single mutants were sensitive towards cell-wall destabilizing agents, suggesting a role in cell-wall assembly or maintenance. Simultaneous deletion of SCW4 and SCW10 showed a synergistic effect, and activated the cell-wall compensatory mechanism in a PKC1-dependent manner. Both the amount of cell-wall chitin and the amount of mannoproteins attached to chitin were increased in mutant scw4scw10. Deletion of CHS3 proved the critical role of chitin in scw4scw10. However, the mannoprotein Sed1p and the glucan synthase Fks2p were also crucial for cell-wall stability in mutant scw4scw10. The exchange of two conserved glutamate residues localized in the putative catalytic domain of GH17 family members strongly suggests that Scw10p acts as a 1,3-β-glucanase or as a 1,3-β-glucanosyltransferase. In addition, the synthetic interactions between Bgl2p and Scw10p which support a functional cooperation in cell-wall assembly were analysed. The data suggest that Scw4p and Scw10p act as glucanases or transglucosidases in concert with other cell-wall proteins to assure cell-wall integrity.
Pubmed 
Willer T, Prados B, Falcón-Pérez JM, Renner-Müller I, Przemeck GK, Lommel M, Coloma A, Valero MC, de Angelis MH, Tanner W, Wolf E, Strahl S, Cruces J. (2004). Targeted disruption of the Walker-Warburg syndrome gene Pomt1 in mouse results in embryonic lethality. Proc Natl Acad Sci U S A. 101(39):14126-31.
Abstract
O-mannosylation is an important protein modification in eukaryotes that is initiated by an evolutionarily conserved family of protein O-mannosyltransferases. The first mammalian protein O-mannosyltransferase gene described was the human POMT1. Mutations in the hPOMT1 gene are responsible for Walker-Warburg syndrome (WWS), a severe recessive congenital muscular dystrophy associated with defects in neuronal migration that produce complex brain and eye abnormalities. During embryogenesis, the murine Pomt1 gene is prominently expressed in the neural tube, the developing eye, and the mesenchyme. These sites of expression correlate with those in which the main tissue alterations are observed in WWS patients. We have inactivated a Pomt1 allele by gene targeting in embryonic stem cells and produced chimeras transmitting the defect allele to offspring. Although heterozygous mice were viable and fertile, the total absence of Pomt1(-/-) pups in the progeny of heterozygous intercrosses indicated that this genotype is embryonic lethal. An analysis of the mutant phenotype revealed that homozygous Pomt1(-/-) mice suffer developmental arrest around embryonic day (E) 7.5 and die between E7.5 and E9.5. The Pomt1(-/-) embryos present defects in the formation of Reichert's membrane, the first basement membrane to form in the embryo. The failure of this membrane to form appears to be the result of abnormal glycosylation and maturation of dystroglycan that may impair recruitment of laminin, a structural component required for the formation of Reichert's membrane in rodents. The targeted disruption of mPomt1 represents an example of an engineered deletion of a known glycosyltransferase involved in O-mannosyl glycan synthesis.
Pubmed 
Hagen I, Ecker M, Lagorce A, Francois JM, Sestak S, Rachel R, Grossmann G, Hauser NC, Hoheisel JD, Tanner W, Strahl S. (2004). Sed1p and Srl1p are required to compensate for cell wall instability in Saccharomyces cerevisiae mutants defective in multiple GPI-anchored mannoproteins. Mol Microbiol. 52(5):1413-25.
Abstract
The covalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI-anchored protein (V. Mrsa et al., 1999, J Bacteriol 181: 3076-3086). Although only 121 amino acids long, the haemagglutinin-tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of > 300 kDa. A membrane-bound form with an apparent molecular mass of 58 kDa is highly O- and N-glycosylated and contains the GPI anchor. With a half-life of 2 min, the membrane form is transformed to the > 300 kDa form. The deletion mutant ccw12Δ grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin. Further, compared with wild-type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol. Interestingly, these defects become less pronounced when further GPI-anchored cell wall proteins are deleted. Mutant DeltaGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) is similar in many respects to wild-type yeast. To find out how the cell wall is stabilized in mutant DeltaGPI, a genome-wide transcription analysis was performed. Of 159 significantly regulated genes, 14 encode either known or suspected cell wall-associated proteins. Analysis of genes affected in transcription revealed that SED1 and SRL1 in particular are required to reconstruct cell wall stability in the absence of multiple GPI-anchored mannoproteins.
Pubmed 
Lommel M, Bagnat M, Strahl S. (2004). Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants. Mol Cell Biol. 24(1):46-57.
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 pmt2pmt4Δ mutant. We found that pmt2pmt4Δ 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 pmt2pmt4Δ mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2pmt4Δ 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.
Pubmed 
Willer T, Valero MC, Tanner W, Cruces J, Strahl S. (2003). O-mannosyl glycans: from yeast to novel associations with human disease. Curr Opin Struct Biol. 13(5):621-30.
Abstract
In yeasts and other fungi, O-mannosyl glycans constitute a major protein modification that is essential for cell viability. For several decades, protein O-mannosylation was considered a yeast-specific modification. Thus, it was especially interesting when it became evident that O-mannosyl glycans in mammals are not as rare as previously thought. O-mannosyl glycans are abundant in the mammalian brain and are also an abundant modification of alpha-dystroglycan, a component of the dystrophin-glycoprotein complex. Recently, mutations in genes that are or might be involved in the glycosylation of alpha-dystroglycan have been identified. Their association with neuromuscular diseases has focused the attention of different research areas on protein O-mannosylation.
Pubmed 
Ecker M, Mrsa V, Hagen I, Deutzmann R, Strahl S, Tanner W. (2003). O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein. EMBO Rep. 4(6):628-32.
Abstract
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.
Pubmed 
Girrbach V, Strahl S. (2003). Members of the evolutionarily conserved PMT family of protein O-mannosyltransferases form distinct protein complexes among themselves. J Biol Chem. 278(14):12554-62.
Abstract
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 of O-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 protein O-mannosylation in fungi.
Pubmed 
Girrbach V, Zeller T, Priesmeier M, Strahl-Bolsinger S. (2000). Structure-function analysis of the dolichyl phosphate-mannose: protein O-mannosyltransferase ScPmt1p. J Biol Chem. 275(25):19288-96.
Abstract
Protein O-mannosylation is an essential protein modification. It is initiated at the endoplasmic reticulum by a family of dolichyl phosphate-mannose:protein O-mannosyltransferases (Pmts), which is evolutionarily conserved from yeast to humans. Saccharomyces cerevisiae Pmt1p is an integral membrane protein of the endoplasmic reticulum. ScPmt1p forms a complex with ScPmt2p that is required for maximum transferase activity. Recently, we proposed a seven-transmembrane structural model for ScPmt1p. A large, hydrophilic, endoplasmic reticulum-oriented segment is flanked by five amino-terminal and two carboxyl-terminal membrane-spanning domains. Based on this model, a structure-function analysis of ScPmt1p was performed. Deletion mutagenesis identified the N-terminal third of the transferase as being essential for the formation of a functional ScPmt1p-ScPmt2p complex. Deletion of the central hydrophilic loop eliminates mannosyltransferase activity, but not ScPmt1p-ScPmt2p interactions. Alignment of all fully characterized PMT family members revealed that this central loop region contains three highly conserved peptide motifs, which can be considered as signatures of the PMT family. In addition, a number of invariant amino acid residues were identified throughout the entire protein sequence. In order to evaluate the functional significance of these conserved residues site-directed mutagenesis was performed. We show that several amino acid substitutions in the conserved motifs significantly reduce ScPmt1p activity. Further, the invariant residues Arg-64, Glu-78, Arg-138, and Leu-408 are essential for ScPmt1p function. In particular, Arg-138 is crucial for ScPmt1p-ScPmt2p complex formation.
Pubmed 
Mrsa V, Ecker M, Strahl-Bolsinger S, Nimtz M, Lehle L, Tanner W. (1999). Deletion of new covalently linked cell wall glycoproteins alters the electrophoretic mobility of phosphorylated wall components of Saccharomyces cerevisiae. J Bacteriol. 181(10):3076-86.
Abstract
The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied. 33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N-glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.
Pubmed 
Strahl-Bolsinger S, Scheinost A. (1999). Transmembrane topology of pmt1p, a member of an evolutionarily conserved family of protein O-mannosyltransferases. J Biol Chem. 274(13):9068-75.
Abstract
The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes. Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino- and carboxyl-terminal sequences containing several putative transmembrane helices. This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far. Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning. Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum. A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function of Pmt1p.
Pubmed 
Strahl-Bolsinger S, Gentzsch M, Tanner W. (1999). Protein O-mannosylation. Biochim Biophys Acta. 1426(2):297-307.
Abstract
Protein O-mannosylation, originally observed in fungi, starts at the endoplasmic reticulum with the transfer of mannose from dolichyl activated mannose to seryl or threonyl residues of secretory proteins. This reaction is catalyzed by a family of protein O-mannosyltransferases (PMTs), which were first characterized in Saccharomyces cerevisiae. The identification of this evolutionarily conserved PMT gene family has led to the finding that protein O-mannosylation plays an essential role in a number of physiologically important processes. Focusing on the PMT gene family, we discuss here the main aspects of the biogenesis of O-linked carbohydrate chains in S. cerevisiae, Candida albicans, and other fungi. We summarize recent work utilizing pmt mutants that demonstrates the impact of protein O-mannosylation on protein secretion, on maintenance of cell wall integrity, and on budding. Further, the occurrence of PMT orthologs in higher eukaryotes such as Arabidopsis, Drosophila and mammals is reported and discussed.
Pubmed 
Strahl-Bolsinger S, Hecht A, Luo K, Grunstein M. (1997). SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev. 11(1):83-93.
Abstract
Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.
Pubmed 
Hecht A, Strahl-Bolsinger S, Grunstein M. (1996). Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature. 383(6595):92-6.
Abstract
Telomeric genes and the HM loci in saccharomyces cerevisiae are transcriptionally repressed and adopt a heterochromatin-like structure. The trans-acting factors RAP1, SIR3 and SIR4 are required for telomeric and HM silencing, and are thought to be chromosomal, but how they contribute to histone-dependent repression of adjacent chromatin is unclear. SIR3 suppresses silencing defects in histones, is limiting for silencing adjacent to telomeres, and interacts with the H3 and H4 amino termini in vitro. Here we show that SIR3 co-immunoprecipitates SIR4, RAP1 and histones from cellular extracts, suggesting the presence of large chromatin-associated protein complexes. Crosslinking experiments show that SIR3 is present at HMRa, HMLalpha and telomeres in vivo, and that is spreads from telomeric regions into adjacent chromatin when overexpressed. Thus SIR3 is a structural component of yeast heterochromatin, repressing adjacent genes as it spreads along the chromosome.
Pubmed 
Hecht A, Laroche T, Strahl-Bolsinger S, Gasser SM, Grunstein M. (1995). Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast. Cell. 80(4):583-92.
Abstract
The silent mating loci and chromosomal regions adjacent to telomeres of S. cerevisiae have features similar to heterochromatin of more complex eukaryotes. Transcriptional repression at these sites depends on the silent information regulators SIR3 and SIR4 as well as histones H3 and H4. We show here that the SIR3 and SIR4 proteins interact with specific silencing domains of the H3 and H4 N-termini in vitro. Certain mutations in these factors, which affect their silencing functions in vivo, also disrupt their interactions in vitro. Immunofluorescence studies with antibodies against RAP1 and SIR3 demonstrate that the H3 and H4 N-termini are required for the association of SIR3 with telomeric chromatin and the perinuclear positioning of yeast telomeres. Based on these interactions, we propose a model for heterochromatin-mediated transcriptional silencing in yeast, which may serve as a paradigm for other eukaryotic organisms as well.
Pubmed 
Grunstein M, Hecht A, Fisher-Adams G, Wan J, Mann RK, Strahl-Bolsinger S, Laroche T, Gasser S. ( 1995). The regulation of euchromatin and heterochromatin by histones in yeast.  J Cell Sci Suppl. 1995;19:29-36.
Abstract
Yeast chromosomes may lack the linker histone H1 (normally required to compact 10 nm beads-on-a-string fiber into the 30 nm fiber) and there is no cytological evidence for higher order fiber structure but they do contain regions which correspond to euchromatin and heterochromatin of higher eukaryotes. Both euchromatin and heterochromatin contain nucleosomal particles (composed of two molecules each of H2A, H2B, H3 and H4), however histones have been shown to regulate genes in these regions in quite different ways. The mechanisms by which such regulation occurs are the topic of this paper.
Pubmed 

Gentzsch M, Strahl-Bolsinger S, Tanner W. (1995). A new Dol-P-Man:protein O-D-mannosyltransferase activity from Saccharomyces cerevisiae. Glycobiology. 5(1):77-82.
Abstract
The deletion of the protein mannosyltransferase 1 gene (PMT1) of Saccharomyces cerevisiae results in viable cells. O-Mannosylation of proteins is reduced to about half of the value in comparison to wild-type cells. In order to distinguish between the the PMT1 gene product (= Pmt1p) and residual transferase activity, an in vitro assay to measure Dol-P-Man:protein mannosyltransferase activity in cells deleted for PMT1 has been developed. The transferase activity of these cells exhibits a pH optimum of 6.5 as compared to pH 7.5 for Pmt1p. The Km value of the residual enzyme activity for the hexapeptide YNPTSV is 7 times higher than that of Pmt1p and shows a clear preference for the seryl residue. Differences in substrate affinities as well as in seryl/threonyl depend on the specific sequence of the peptides used in the enzyme assay. The new enzyme activity shows a significantly lower thermal stability as compared to Pmt1p.
Pubmed 
Tanner W, Gentzsch M, Immervoll T, Scheinost A, Strahl-Bolsinger S. (1995). Fungal glycoproteins and their biosynthetic pathway as potential targets for antifungal agents. 7. Acta Biochim Pol. 1995;42(4):505-8.
Abstract
The yeast cell wall as a good antifungal target is discussed in general. More specifically the reaction, catalyzed by Dol-P-Man: protein O-D-mannosyltransferase is proposed as a new potential target. Six genes responsible for this endoplasmic reticulum-localized reaction have been cloned and characterized so far. Triple disruptions of these genes are either lethal or the corresponding cells have to be osmotically stabilized to survive. No inhibitors of this reaction are as yet known.
Pubmed 
Strahl-Bolsinger S, Immervoll T, Deutzmann R, Tanner W. (1993). PMT1, the gene for a key enzyme of protein O-glycosylation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 90(17):8164-8.
Abstract
The integral endoplasmic reticulum membrane protein catalyzing the initial reaction of protein O-glycosylation in Saccharomyces cerevisiae has been purified to homogeneity. The 92-kDa N-glycosylated protein transfers mannose residues from dolichyl phosphate-D-mannose to specific serine/threonine residues of proteins entering the secretory pathway. This type of mannosyl transfer reaction has so far been observed only in fungal cells. Oligonucleotides derived from peptide sequences of the transferase were used to screen a genomic yeast library. A clone was isolated which contains an open reading frame of 2451 bp corresponding to an mRNA transcript of 3 kb. The predicted protein consists of 817 amino acids including three potential N-glycosylation sites. The hydropathy plot indicates a tripartite structure of the protein: an amino-terminal third and a carboxyl-terminal third, both with multiple potential transmembrane helices, and a central hydrophilic part. Expression of the clone in Escherichia coli resulted in mannosyltransferase activity. Gene disruption led to a complete loss of in vitro mannosyltransferase activity from dolichyl phosphate-D-mannose to a peptide used as acceptor in the enzymatic assay. In vivo it was observed, however, that protein O-mannosylation in the disruptant had decreased only to about 40-50%, indicating the existence of an additional transferase which had not been measured by the in vitro enzyme assay.
Pubmed 
Strahl-Bolsinger S, Tanner W. (1993). A yeast gene encoding a putative RNA helicase of the "DEAD"-box family. Yeast. 9(4):429-32.
Abstract
An unknown open reading frame from Saccharomyces cerevisiae was identified and sequenced. The predicted amino acid sequence shows high homology to the DEAD-box family of proteins. Gene disruption revealed that the gene is not essential for yeast but necessary for normal cell growth.
Pubmed 
Strahl-Bolsinger S, Tanner W. (1991). Protein O-glycosylation in Saccharomyces cerevisiae. Purification and characterization of the dolichyl-phosphate-D-mannose-protein O-D-mannosyltransferase. Eur J Biochem. 196(1):185-90.
Abstract
The enzyme dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides. The specific activity of the protein was enriched 130-fold before it was further purified by native and SDS gel chromatography. A 92-kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase. The 92-kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains. The purified mannosyltransferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides. Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino-terminal and carboxy-terminal neighbours, respectively.
Pubmed 

 


/var/www/cos/ / http://www.cos.uni-heidelberg.de/ Prof. Dr. Sabine Strahl _e