Protein O-mannosylation Research Articles

The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

Effect of Protein O-Mannosyltransferase (MSMEG_5447) on M. smegmatis and Its Survival in Macrophages.

Protein O-mannosyltransferase (PMT) catalyzes an initial step of protein O-mannosylation of Mycobacterium tuberculosis (Mtb) and plays a crucial role for Mtb survival in the host. To better understand the role of PMT in the host innate immune response during mycobacterial infection, in this study, we utilized Mycobacterium smegmatis pmt (MSMEG_5447) gene knockout strain, ΔM5447, to infect THP-1 cells. Our results revealed that the lack of MSMEG_5447 not only impaired the growth of M. smegmatis in 7H9 medium but also reduced the resistance of M. smegmatis against lysozyme and acidic stress in vitro. Macrophage infection assay showed that ΔM5447 displayed attenuated growth in macrophages at 24 h post-infection. The production of TNF-α and IL-6 and the activation of transcription factor NF-κB were decreased in ΔM5447-infected macrophages, which were further confirmed by transcriptomic analysis. Moreover, ΔM5447 failed to inhibit phagosome–lysosome fusion in macrophages. These findings revealed that PMT played a role in modulating the innate immune responses of the host, which broaden our understanding for functions of protein O-mannosylation in mycobacterium–host interaction.

Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell–cell adhesion

Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle–eye–brain (MEB) disease. In addition to defects in cell–extracellular matrix adhesion, aberrant cell–cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell–cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell–cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial–mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell–cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated.

Functional implications of MIR domains in protein O-mannosylation.

Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a β-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general β-trefoil carbohydrate-binding sites (α, β), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous β-trefoil - carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.

Protein O-mannosylation affects protein secretion, cell wall integrity and morphogenesis in Trichoderma reesei

Protein O-mannosyltransferases (PMTs) initiate O-mannosylation of proteins in the ER. Trichoderma reesei strains displayed a single representative of each PMT subfamily, Trpmt1, Trpmt2 and Trpmt4. In this work, two knockout strains ΔTrpmt1and ΔTrpmt4were obtained. Both mutants showed retarded growth, defective cell walls, reduced conidiation and decreased protein secretion. Additionally, the ΔTrpmt1strain displayed a thermosensitive growth phenotype, while the ΔTrpmt4 strain showed abnormal polarity. Meanwhile, OETrpmt2 strain, in which the Trpmt2 was over-expressed, exhibited increased conidiation, enhanced protein secretion and abnormal polarity. Using a lectin enrichment method and MS/MS analysis, 173 O-glycoproteins, 295 O-glycopeptides and 649 O-mannosylation sites were identified as the targets of PMTs in T. reesei. These identified O-mannoproteins are involved in various physiological processes such as protein folding, sorting, transport, quality control and secretion, as well as cell wall integrity and polarity. By comparing proteins identified in the mutants and its parent strain, the potential specific protein substrates of PMTs were identified. Based on our results, TrPMT1 is specifically involved inO-mannosylation of intracellular soluble proteins and secreted proteins, specially glycosidases. TrPMT2 is involved inO-mannosylation of secreted proteins and GPI-anchor proteins, and TrPMT4 mainly modifies multiple transmembrane proteins. The TrPMT1-TrPMT4 complex is responsible for O-mannosylation of proteins involved in cell wall integrity. Overexpression of TrPMT2 enhances protein secretion, which might be a new strategy to improve expression efficiency in T. reesei.

Translational Regulation of Pmt1 and Pmt2 by Bfr1 Affects Unfolded Protein O-Mannosylation.

O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding green fluorescent protein (GFP) as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Structure of the eukaryotic protein O-mannosyltransferase Pmt1-Pmt2 complex.

In eukaryotes, a nascent peptide entering the endoplasmic reticulum (ER) is scanned by two Sec61-translocon-associated large membrane machines for protein N-glycosylation and protein O-mannosylation, respectively. While the structure of the eight-protein oligosaccharyltransferase complex has been determined recently, the structures of mannosyltransferases of the PMT family, which are an integral part of ER protein homeostasis, are still unknown. Here we report cryo-EM structures of the S. cerevisiae Pmt1–Pmt2 complex bound to a donor and an acceptor peptide at 3.2-Å resolution, showing that each subunit contains 11 transmembrane helices and a lumenal β-trefoil fold termed the MIR domain. The structures reveal the substrate recognition model and confirm an inverting mannosyl-transferring reaction mechanism by the enzyme complex. Furthermore, we found that the transmembrane domains of Pmt1 and Pmt2 share a structural fold with the catalytic subunits of oligosaccharyltransferases, confirming a previously proposed evolutionary relationship between protein O-mannosylation and protein N-glycosylation.

Identification of essential residues for polyprenol phosphate mannose synthase and protein O-mannosyl transferase activities required for protein glycosylation in Streptomyces

Actinobacteria have a protein O-glycosylation system that resembles eukaryotic protein O-mannosylation. Both M. tuberculosis and S. coelicolor have growth retarded phenotypes when protein-O-mannosyl transferase (Pmt), which transfers mannose from polyprenol phosphate mannose to a target protein, is absent. Moreover, S. coelicolor pmt- mutants are resistant to φC31 phage infection and have increased susceptibility to vancomycin and multiple b-lactams. S. coelicolor strains that lack polyprenol phosphate mannose synthase (Ppm1), which transfers mannose from GDP-mannose to polyprenol phosphate, are even more susceptible to antibiotics and a ppm1- mutant in M. tuberculosis is lethal. Pmt and Ppm1 are therefore possible new targets for the isolation of antimicrobials to be used against M. tuberculosis. Our aim is to further understand the structure and function of these enzymes. Sequence alignments and structural bioinformatics were used for S. coelicolor Ppm1 and Pmt to identify site-directed mutagenesis targets. Mutant alleles were introduced into ppm1- (DT3017) or pmt- (DT2008) S. coelicolor strains using conjugative integrative plasmids and scored for their ability to complement phage sensitivity and antibiotic hyper-susceptible phenotypes. Twenty-two highly conserved Pmt residues were each changed to alanine and six mutants failed to complement DT2008, indicating essentiality. Modelling these six residues indicated that five are positioned close to the predicted catalytic DE motif. For Ppm1, ten mutant alleles were tested and eight were essential for DT3017 complementation, with four residues positioned close to the predicted catalytic DXD motif. Whilst some of the mutations were predicted to impair catalytic activity, others may have affected localisation or substrate binding.

Molecular genetics of the POMT1-related muscular dystrophy-dystroglycanopathies.

Protein O-mannosyltransferase 1 (POMT1) is a critical enzyme participating in the first step of protein O-mannosylation. Mutations in the coding gene, POMT1, have been described to be related to a series of autosomal recessive disorders associated with defective alpha-dystroglycan glycosylation, later termed muscular dystrophy-dystroglycanopathies (MDDGs). MDDGs are characterized by a broad phenotypic spectrum of congenital muscular dystrophy or later-onset limb-girdle muscular dystrophy, accompanied by variable degrees of intellectual disability, brain defects, and ocular abnormalities. To date, at least 76 disease-associated mutations in the POMT1 gene, including missense, nonsense, splicing, deletion, insertion/duplication, and insertion-deletion mutations, have been reported in the literature. In this review, we highlight the present knowledge of the identified disease-associated POMT1 gene mutations and genetic animal models related to the POMT1 gene. This review may help further normative classification of phenotypes, assist in definite clinical and genetic diagnoses, and genetic counseling, and may comprehensively improve our understanding of the basis of complex phenotypes and possible pathogenic mechanisms involved.

Genotype‐Phenotype Correlations for Protein O‐Linked Mannose NAcetylglucosaminyltransferase 1 in Congenital Muscular Dystrophy

Congenital muscular dystrophy (CMD) is a heterogeneous family of inherited muscle disorders. A subtype of CMD known as dystroglycanopathy is classified by hypoglycosylation of alpha‐dystroglycan (α‐DG). α‐DG, an integral component of the dystrophin‐glycoprotein complex, is responsible for connecting the actin cytoskeleton to the extracellular matrix in muscle tissues. Hypoglycosylation of α‐DG arises from defects in the protein O‐mannosylation biosynthetic pathway. One enzyme involved in the O‐mannosylation pathway is protein O‐linked‐mannose β‐1,2‐N‐acetylglucosaminyltransferase (POMGNT1). On serine and threonine residues in α‐DG, POMGNT1 catalyzes addition of N‐acetyl‐D‐glucosamine (GlcNAc) to an O‐mannose structure in a β1,2‐linkage to create a core m1 glycan structure in the cis‐Golgi. Mutations in the gene encoding POMGNT1 have been observed in patients with various forms of dystroglycanopathy.Our work examines the role of R311Q, P493R, T176P, P303L, C269Y, E223K, C490Y, G502A, R605P, I287S, E156K, D395N, and D556N mutations in POMGNT1 to define a genotype‐phenotype correlation. The POMGNT1 mutations present a range of clinical presentations, from muscle‐eye‐brain disease, an extremely severe condition characterized by muscle weakness, vision and brain abnormalities, and developmental disabilities, to limb‐girdle muscular dystrophy, a much milder phenotype that results in progressive muscle weakness and wasting but lacks neurological comorbidities. The effects of the selected mutations on enzyme characteristics are not well established in the literature. Therefore, we sought to identify mutation‐derived changes in enzyme kinetics and stability.To do so, HEK293F cells were transfected with mutant plasmids generated by site‐directed mutagenesis. Preliminary data indicated that R311Q, D395N, R605P, and D556N POMGNT1 mutants maintained enzyme expression. Radiolabel transfer assays established that the R311Q, D395N, and R605P mutants were kinetically dead while the D556N mutant still exhibited transfer. Promega's UDP‐Glo™ assay was performed on the D556N mutant, which demonstrated reduced kinetic activity compared to wild‐type POMGNT1. A SYPRO Orange thermal shift assay revealed the aforementioned POMGNT1 mutants to be thermodynamically stable. We are currently experimenting with the rest of the mutants and are also investigating the ability of all mutants to rescue POMGNT1 knockout cell lines for IIH6 reactivity, laminin binding, and Lassa pseudovirus entry. Understanding genotype‐phenotype correlations in those glycosyltransferases will facilitate the design of more targeted treatments for individuals based on the mutation(s) they carry.Support or Funding InformationThis work was supported in part by NIGMS/NIH (R01GM111939, LW).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics.

Polyprenol phosphate mannose (PPM) is a lipid-linked sugar donor used by extra-cytoplasmic glycosyl tranferases in bacteria. PPM is synthesiszed by polyprenol phosphate mannose synthase, Ppm1, and in most Actinobacteria is used as the sugar donor for protein O-mannosyl transferase, Pmt, in protein glycosylation. Ppm1 and Pmt have homologues in yeasts and humans, where they are required for protein O-mannosylation. Actinobacteria also use PPM for lipoglycan biosynthesis. Here we show that ppm1 mutants of Streptomyces coelicolor have increased susceptibility to a number of antibiotics that target cell wall biosynthesis. The pmt mutants also have mildly increased antibiotic susceptibilities, in particular to β-lactams and vancomycin. Despite normal induction of the vancomycin gene cluster, vanSRJKHAX, the pmt and ppm1 mutants remained highly vancomycin sensitive indicating that the mechanism of resistance is blocked post-transcriptionally. Differential RNA expression analysis indicated that catabolic pathways were downregulated and anabolic ones upregulated in the ppm1 mutant compared to the parent or complemented strains. Of note was the increase in expression of fatty acid biosynthetic genes in the ppm1- mutant. A change in lipid composition was confirmed using Raman spectroscopy, which showed that the ppm1- mutant had a greater relative proportion of unsaturated fatty acids compared to the parent or the complemented mutant. Taken together, these data suggest that an inability to synthesize PPM (ppm1) and loss of the glycoproteome (pmt- mutant) can detrimentally affect membrane or cell envelope functions leading to loss of intrinsic and, in the case of vancomycin, acquired antibiotic resistance.

Protein O-Mannosyltransferases Affect Sensory Axon Wiring and Dynamic Chirality of Body Posture in the Drosophila Embryo.

Genetic defects in protein O-mannosyltransferase 1 (POMT1) and POMT2 underlie severe muscular dystrophies. POMT genes are evolutionarily conserved in metazoan organisms. In Drosophila, both male and female POMT mutants show a clockwise rotation of adult abdominal segments, suggesting a chirality of underlying pathogenic mechanisms. Here we described and analyzed a similar phenotype in POMT mutant embryos that shows left-handed body torsion. Our experiments demonstrated that coordinated muscle contraction waves are associated with asymmetric embryo rolling, unveiling a new chirality marker in Drosophila development. Using genetic and live-imaging approaches, we revealed that the torsion phenotype results from differential rolling and aberrant patterning of peristaltic waves of muscle contractions. Our results demonstrated that peripheral sensory neurons are required for normal contractions that prevent the accumulation of torsion. We found that POMT mutants show abnormal axonal connections of sensory neurons. POMT transgenic expression limited to sensory neurons significantly rescued the torsion phenotype, axonal connectivity defects, and abnormal contractions in POMT mutant embryos. Together, our data suggested that protein O-mannosylation is required for normal sensory feedback to control coordinated muscle contractions and body posture. This mechanism may shed light on analogous functions of POMT genes in mammals and help to elucidate the etiology of neurological defects in muscular dystrophies.SIGNIFICANCE STATEMENT Protein O-mannosyltransferases (POMTs) are evolutionarily conserved in metazoans. Mutations in POMTs cause severe muscular dystrophies associated with pronounced neurological defects. However, neurological functions of POMTs remain poorly understood. We demonstrated that POMT mutations in Drosophila result in abnormal muscle contractions and cause embryo torsion. Our experiments uncovered a chirality of embryo movements and a unique POMT-dependent mechanism that maintains symmetry of a developing system affected by chiral forces. Furthermore, POMTs were found to be required for proper axon connectivity of sensory neurons, suggesting that O-mannosylation regulates the sensory feedback controlling muscle contractions. This novel POMT function in the peripheral nervous system may shed light on analogous functions in mammals and help to elucidate pathomechanisms of neurological abnormalities in muscular dystrophies.

Protein O-mannosylation deficiency increases LprG-associated lipoarabinomannan release by Mycobacterium tuberculosis and enhances the TLR2-associated inflammatory response

Protein O-mannosylation is crucial for the biology of Mycobacterium tuberculosis but the key mannosylated protein(s) involved and its(their) underlying function(s) remain unknown. Here, we demonstrated that the M. tuberculosis mutant (Δpmt) deficient for protein O-mannosylation exhibits enhanced release of lipoarabinomannan (LAM) in a complex with LprG, a lipoprotein required for LAM translocation to the cell surface. We determined that LprG is O-mannosylated at a unique threonine position by mass spectrometry analyses of the purified protein. However, although replacement of this amino acid by an alanine residue completely abolished LprG O-mannosylation, the increased release of the LAM/LprG complex was preserved. We found that the increased secretion of this complex is due to enhanced LAM production in the Δpmt M. tuberculosis and M. smegmatis mutants relative to their wild-type counterparts. This abnormal release of LAM/LprG has functional consequences on the induction of inflammatory responses and provides a possible explanation for the reduced virulence of the M. tuberculosis Δpmt mutant.

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated α-dystroglycanopathies, because deficient O-Man glycosylation of α-dystroglycan disrupts laminin interaction with α-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose β-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to α-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to α-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins.

Whoa man! Unexpected protein O-mannosylation pathways in mammals

The recent expansion of well-characterized O-mannosylated mammalian proteins beyond the archetypical example of α-dystroglycan has inspired new interest in the possibility of additional functional roles of this modification. In an effort to explore those roles, a new study now serendipitously uncovers the existence of an alternative pathway to the well-described POMT (protein O-mannosyltransferase) family of O-mannosyltransferases.

Cellular Consequences of Diminished Protein O-Mannosyltransferase Activity in Baker's Yeast.

O-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein O-mannosyltransferase (PMT) family. Despite the vital role of O-mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein O-mannosylation, we performed a genome-wide screen to identify Saccharomyces cerevisiae mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a. We identified the cell wall and the ER as the cell compartments affected most upon PMT inhibition. Especially mutants with defects in N-glycosylation, biosynthesis of glycosylphosphatidylinositol-anchored proteins and cell wall β-1,6-glucan showed impaired growth when O-mannosylation became limiting. Signaling pathways that counteract cell wall defects and unbalanced ER homeostasis, namely the cell wall integrity pathway and the unfolded protein response, were highly crucial for the cell growth. Moreover, among the most affected mutants, we identified Ost3, one of two homologous subunits of the oligosaccharyltransferase complexes involved in N-glycosylation, suggesting a functional link between the two pathways. Indeed, we identified Pmt2 as a substrate for Ost3 suggesting that the reduced function of Pmt2 in the absence of N-glycosylation promoted sensitivity to the drug. Interestingly, even though S. cerevisiae Pmt1 and Pmt2 proteins are highly similar on the sequence, as well as the structural level and act as a complex, we identified only Pmt2, but not Pmt1, as an Ost3-specific substrate protein.

Induction of Antibodies Directed Against Branched Core O-Mannosyl Glycopeptides-Selectivity Complimentary to the ConA Lectin.

Mammalian protein O-mannosylation, initiated by attachment of α-mannopyranose to Ser or Thr residues, comprise a group of post-translational modifications (PTMs) involved in muscle and brain development. Recent advances in glycoproteomics methodology and the "SimpleCell" strategy have enabled rapid identification of glycoproteins and specific glycosylation sites. Despite the enormous progress made, the biological impact of the mammalian O-mannosyl glycoproteome remains largely unknown to date. Tools are still needed to investigate the structure, role, and abundance of O-mannosyl glycans. Although O-mannosyl branching has been shown to be of relevance in integrin-dependent cell migration, and also plays a role in demyelinating diseases, such as multiple sclerosis, a broader understanding of the biological roles of branched O-mannosyl glycans is lacking in part due to the paucity of detection tools. In this work, a glycopeptide vaccine construct was synthesized and used to generate antibodies against branched O-mannosyl glycans. Glycopeptide microarray screening revealed high selectivity of the induced antibodies for branched glycan core structures presented on different peptide backbones, with no cross-reactivity observed with related linear glycans. For comparison, microarray screening of the mannose-binding lectin concanavalin A (ConA), which is commonly used in glycoproteomics workflows to enrich tryptic O-mannosyl peptides, showed that the ConA lectin did not recognize branched O-mannosyl glycans. The binding preference of ConA for short linear O-mannosyl glycans was rationalized in terms of molecular structure using crystallographic data augmented by molecular modeling. The contrast between the ConA binding specificity and that of the new antibodies indicates a novel role for the antibodies in studies of protein O-mannosylation.

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.

Dom34 Links Translation to Protein O-mannosylation.

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5′-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5′-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3′-UTR of transcripts.