Mark Lommel

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.

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 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.

Protein N‐glycosylation determines functionality of the Saccharomyces cerevisiae cell wall integrity sensor Mid2p

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.

O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues.

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.

POMT2, a key enzyme in Walker-Warburg syndrome: somatic sPOMT2, but not testis-specific tPOMT2, is crucial for mannosyltransferase activity in vivo

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.

POMT1 is Essential for Protein O-Mannosylation in Mammals

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.

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

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.

Genetic knockdown and knockout approaches in Hydra

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.