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Research background. Glycosyltransferases represent a large and diverse family of enzymes that catalyze the transfer of sugar residues to proteins and lipids, thereby regulating essential cellular processes such as protein quality control and cell wall biosynthesis. In yeast, protein O-mannosyltransferases and other glycosyltransferases are crucial for maintaining cell wall integrity. While the functions of many of these enzymes are well characterized, the role of some of them, such as Gmh5p, remains unknown. This study aims to elucidate the function of Gmh5p, a previously uncharacterized member of the GT34 glycosyltransferase family, in the context of protein and cell wall biosynthesis in Schizosaccharomyces pombe. Experimental approach. To identify proteins and pathways compensating for reduced O-mannosylation, we performed a genetic screening for multi-copy suppressors in a conditional lethal nmt81-oma2+ mutant background. The enzymatic activity of Gmh5p was biochemically characterized, and its functional homology to known mannosyltransferases was assessed through complementation experiments in Saccharomyces cerevisiae. In addition, the N-glycosylation status of model substrates was analyzed in gmh5Δ mutant strains. Results and conclusions. Gmh5p was identified as a suppressor of O-mannosylation defects. Contrary to its predicted function, Gmh5p did not exhibit α-1,2-galactosyltransferase activity but instead showed mannosyltransferase activity. Expression of gmh5+ in S. cerevisiae mnn10 mutants restored hygromycin tolerance to near wild-type levels. Furthermore, N-glycosylation of model substrates was reduced in gmh5Δ mutants. These results demonstrate that Gmh5p is a mannosyltransferase involved in the outer chain elongation of N-linked glycans and functions as a homologue of Mnn10p. Novelty and scientific contribution. This study provides the first functional characterization of Gmh5p as a mannosyltransferase of the GT34 family and demonstrates its role in N-glycan biosynthesis. Our findings expand the current understanding of the diversity and specificity of glycosyltransferases in eukaryotes and highlight their importance in cell wall biology.

Kevin Campbell and colleagues identify mutations in ISPD as a cause of Walker-Warburg syndrome (WWS) using a complementation assay in fibroblasts derived from affected individuals and targeted sequencing. They find that loss-of-function mutations in ISPD disrupt dystroglycan O-mannosylation, suggesting a new disease mechanism. 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 1 . 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 2 . 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.

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.

Thrombospondins (TSPs) are multidomain glycoproteins with complex matricellular functions in tissue homeostasis and remodeling. We describe a novel role of TSP as a Wnt signaling target in the basal eumetazoan Hydra . Proteome analysis identified Hydra magnipapillata TSP (HmTSP) as a major component of the cnidarian mesoglea. In general, the domain organization of cnidarian TSPs is related to the pentameric TSPs of bilaterians, and in phylogenetic analyses cnidarian TSPs formed a separate clade of high sequence diversity. HmTSP expression in polyps was restricted to the hypostomal tip and tentacle bases that harbor Wnt-regulated organizer tissues. In the hypostome, HmTSP- and Wnt3 -expressing cells were identical or in close vicinity to each other, and regions of ectopic tentacle formation induced by pharmacological β-Catenin activation (Alsterpaullone) corresponded to foci of HmTSP expression. Chromatin immunoprecipitation (ChIP) confirmed binding of Hydra TCF to conserved elements in the HmTSP promotor region. Accordingly, β-Catenin knockdown by siRNAs reduced normal HmTSP expression at the head organizer. In contrast, knockdown of HmTSP expression led to increased numbers of ectopic organizers in Alsterpaullone-treated animals, indicating a negative regulatory function. Our data suggest an unexpected role for HmTSP as a feedback inhibitor of Wnt signaling during Hydra body axis patterning and maintenance.

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.

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.

Experimental approach. To identify proteins and pathways compensating for reduced O-mannosylation, we performed a genetic screening for multicopy suppressors in a conditional lethal nmt81-[oma2.sup.+] mutant background. The enzymatic activity of Gmh5p was biochemically characterized, and its functional homology to known mannosyltransferases was assessed through complementation experiments in Saccharomyces cerevisiae. In addition, the N-glycosylation status of model substrates was analyzed in gmh5[DELTA] mutant strains. Novelty and scientific contribution. This study provides the first functional characterization of Gmh5p as a mannosyltransferase of the GT34 family and demonstrates its role in N-glycan biosynthesis. Our findings expand the current understanding of the diversity and specificity of glycosyltransferases in eukaryotes and highlight their importance in cell wall biology.

Hydra’s almost unlimited regenerative potential is based on Wnt signaling, but so far it is unknown how the injury stimulus is transmitted to discrete patterning fates in head and foot regenerates. We previously identified mitogen-activated protein kinases (MAPKs) among the earliest injury response molecules in Hydra head regeneration. Here, we show that three MAPKs—p38, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs)—are essential to initiate regeneration in Hydra, independent of the wound position. Their activation occurs in response to any injury and requires calcium and reactive oxygen species (ROS) signaling. Phosphorylated MAPKs hereby exhibit cross talk with mutual antagonism between the ERK pathway and stress-induced MAPKs, orchestrating a balance between cell survival and apoptosis. Importantly, Wnt3 and Wnt9/10c, which are induced by MAPK signaling, can partially rescue regeneration in tissues treated with MAPK inhibitors. Also, foot regenerates can be reverted to form head tissue by a pharmacological increase of β-catenin signaling or the application of recombinant Wnts. We propose a model in which a β-catenin-based stable gradient of head-forming capacity along the primary body axis, by differentially integrating an indiscriminate injury response, determines the fate of the regenerating tissue. Hereby, Wnt signaling acquires sustained activation in the head regenerate, while it is transient in the presumptive foot tissue. Given the high level of evolutionary conservation of MAPKs and Wnts, we assume that this mechanism is deeply embedded in our genome.

The findings of this hermeneutical phenomenological study describe how Christian engineering faculty members experience their spirituality (divine-human transformational relationship) and how their discipline and spirituality affect one another. Discoveries resulting from 10 interviews with Christian scholar engineers indicateD they use their discipline to understand God and their spirituality and vice versa. Participants understood their faculty role as working for God and mentoring students beyond the discipline. Spiritual struggles exist when lived experiences do not align with participants’ spiritual understanding. This study provides additional insight into spirituality and faculty members, specifically the spirituality of engineering faculty members. The outcomes of this study may provide institutional leaders ideas to promote connections between the spirituality of engineering faculty members and their discipline.

Hydra is a member of the Cnidaria, an ancient phylum at the base of metazoan evolution and sister group to all bilaterian animals. The regeneration capacity of Hydra, mediated by its stem cell systems, is unparalleled in the animal kingdom. The recent sequencing of the Hydra genome and that of other cnidarians has drawn new attention to this well-known model organism. In spite of this, the establishment of methods to manipulate gene expression in Hydra have remained a major challenge. Here we report a CRISPR-Cas9 based targeted mutation approach as well as an optimized, reproducible strategy for the delivery of siRNAs. Both approaches are based on a refined electroporation protocol for adult Hydra polyps. We demonstrate that these strategies provide reliable genetic interference with target gene expression, facilitating functional studies and genome editing in Hydra.