Walker-Warburg syndrome (WWS) is clinically defined as congenital muscular dystrophy accompanied by a variety of brain and eye malformations. It represents the most severe clinical phenotype in a spectrum of alpha-dystroglycan posttranslational processing abnormalities, which share a defect in laminin binding glycan synthesis1. Although six WWS causing genes have been described, only half of all patients can currently be diagnosed genetically2. A cell fusion complementation assay using fibroblasts from undiagnosed WWS individuals identified five novel complementation groups. Further evaluation of one group by linkage analysis and targeted sequencing identified recessive mutations in the isoprenoid synthase domain containing (ISPD) gene. Confirmation of the pathogenicity of the identified ISPD mutations was demonstrated 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 novel mechanism for WWS pathophysiology.

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.

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.

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.Protein O-mannosylation is an evolutionarily conserved protein modification of fundamental importance in many eukaryotes. In yeasts and fungi, the attachment of O-linked mannosyl residues to proteins of the secretory pathway is essential for cell viability (1). In particular it is indispensable for cell wall integrity and normal cellular morphogenesis (2-4). Impairment of O-mannosylation also affects the stability, localization, and/or proper function of individual proteins (5-10). Furthermore, aberrant O-mannosylation can interfere with the retrograde transport of misfolded proteins across the membrane of the endoplasmic reticulum (ER) 1 (11). O-mannosylation is not only important in yeast, but also in mammals. It was recently shown that in humans, O-mannosyl glycosylation represents a new pathomechanism for muscular dystrophy and neuronal migration disorders (12, 13).In yeast and fungi, O-mannosylation is initiated in the lumen of the ER by an essential family of protein O-mannosyltransferases (PMTs). These enzymes catalyze the transfer of mannose from dolichyl phosphate-activated mannose (Dol-PMan) to serine or threonine residues of secretory proteins (2). In Saccharomyces cerevisiae, a total of seven PMT family members (Pmt1-7p) have been identified, which share almost identical hydropathy profiles that predict the PMTs to b...

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 pmt4⌬ mutant. We found that pmt2 pmt4⌬ 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 pmt4⌬ mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4⌬ 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 initiated at the endoplasmic reticulum (ER) by the transfer of mannose from dolichyl phosphate-activated mannose to serine or threonine residues of secretory proteins (52). This reaction is catalyzed by an essential family of protein O-mannosyltransferases (PMTs) that is evolutionarily conserved from yeast to humans (28,52,57). The PMT family is divided into the PMT1, PMT2, and PMT4 subfamilies, whose members include transferases closely related to Saccharomyces cerevisiae Pmt1p, Pmt2p, and Pmt4p, respectively (17,57). In S. cerevisiae the entire PMT family is highly redundant (Pmt1 to Pmt7p), and members of the PMT1 and PMT2 subfamilies show marked similarities and distinctions from PMT4 subfamily members. For example, members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p), whereas the unique representative of the PMT4 subfamily forms homomeric complexes (16). Further, the PMT1/PMT2 and PMT4 subfamilies use different acceptor protein substrates in vivo (10,14).Studies of pmt mutants revealed that protein O mannosylation plays a substantial role in uni-and multicellular eukaryotes. In humans, mutations in POMT1 (protein O-mannosyltransferase 1 gene), which encodes a putative counterpart of the yeast Pmt4p O-mannosyltransferase, result in Walker-Warburg syndrome, which is characterized by severe congenital muscular dystrophy, a neuronal migration defect, and structural abnormalities of the eye (2). Mutations of the Drosophila POMT1 orth...