Protein tyrosine phosphatase 69D is a substrate of protein O-mannosyltransferases 1-2 that is required for the wiring of sensory axons in Drosophila.

Mutations in protein O-mannosyltransferases (POMTs) result in severe brain defects and congenital muscular dystrophies characterized by abnormal glycosylation of α-dystroglycan (α-Dg). However, neurological phenotypes of POMT mutants are not well understood, and the functional substrates of POMTs other than α-Dg remain unknown. Using a Drosophila model, here we reveal that Dg alone cannot account for the phenotypes of POMT mutants, and identify Protein tyrosine phosphatase 69D (PTP69D) as a gene interacting with POMTs in producing the abdomen rotation phenotype. Using RNAi-mediated knockdown, mutant alleles, and a dominant-negative form of PTP69D, we reveal that PTP69D is required for the wiring of larval sensory axons. We also found that PTP69D and POMT genes interact in this process, and that their interactions lead to complex synergistic or antagonistic effects on axon wiring phenotypes, depending on the mode of genetic manipulation. Using glycoproteomic approaches, we further characterized the glycosylation of the PTP69D transgenic construct expressed in genetic strains with different levels of POMT activity. We found that the PTP69D construct carries many O-linked mannose modifications when expressed in Drosophila with wild-type or ectopically upregulated expression of POMTs. These modifications were absent in POMT mutants, suggesting that PTP69D is a substrate of POMT-mediated O-mannosylation. Taken together, our results indicate that PTP69D is a novel functional substrate of POMTs that is required for axon connectivity. This mechanism of POMT-mediated regulation of receptor-type protein tyrosine phosphatase functions could potentially be conserved in mammals and may shed new light on the etiology of neurological defects in muscular dystrophies.

Development of a novel target-based cell assay, reporter of the activity of Mycobacterium tuberculosis protein-O-mannosyltransferase.

The Protein-O-mannosyltransferase is crucial for the virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This enzyme, called MtPMT (Rv1002c), is responsible for the post-translational O-mannosylation of mycobacterial proteins. It catalyzes the transfer of a single mannose residue from a polyprenol phospho-mannosyl lipidic donor to the hydroxyl groups of selected Ser/Thr residues in acceptor proteins during their translocation across the membrane. Previously, we provided evidence that the loss of MtPMT activity causes the absence of mannoproteins in Mycobacterium tuberculosis, severely impacting its intracellular growth, as well as a strong attenuation of its pathogenicity in immunocompromised mice. Therefore, it is of interest to develop specific inhibitors of this enzyme to better understand mycobacterial infectious diseases. Here we report the development of a "target-based" phenotypic assay for this enzyme, assessing its O-mannosyltransferase activity in bacteria, in the non-pathogenic Mycobacterium smegmatis strain. Robustness of the quantitative contribution of this assay was evaluated by intact protein mass spectrometry, using a panel of control strains, overexpressing the MtPMT gene, carrying different key point-mutations. Then, screening of a limited library of 30 compounds rationally chosen allowed us to identify 2 compounds containing pyrrole analogous rings, as significant inhibitors of MtPMT activity, affecting neither the growth of the mycobacterium nor its secretion of mannoproteins. These molecular cores could therefore serve as scaffold for the design of new pharmaceutical agents that could improve treatment of mycobacterial diseases. We report here the implementation of a miniaturized phenotypic activity assay for a glycosyltransferase of the C superfamily.

O-mannosyltransferase MaPmt2 contributes to stress tolerance, cell wall integrity and virulence in Metarhizium acridum.

As a conserved post-translational modification, O-mannosyltransferase families play important roles in many cellular processes. Three subfamilies (MaPmt1, MaPmt2 and MaPmt4) are grouped in Metarhizium acridum according to sequence homology. The functions of MaPmt1 and MaPmt4 have been characterized in M. acridum previously. In this study, the functions of another member belonging to the Pmt2 subfamily, MaPmt2, were identified through RNAi strategy. The three MaPmt2 knockdown mutants showed dramatically decreased expression of MaPmt2. Phenotypic analyses showed that the mutants exhibited decreased tolerances to wet-heat, UV-B irradiation and cell wall perturbing chemicals. Further studies revealed that the mutants presented thinner cell walls observed by transmission electron microscope combined with changed cell wall components. Besides, knockdown of MaPmt2 decelerated conidial germination and decreased conidial yield. Compared with the wild-type strain, the MaPmt2 knockdown mutants caused impaired virulence only by topical inoculation. Results illustrated that the decreased virulence by inoculation could result from the delayed conidial germination on locust wings, reduced appressorium formation, as well as reduced turgor pressure in MaPmt2 knockdown mutants.

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.

Genetical and O-glycoproteomic analyses reveal the roles of three protein O-mannosyltransferases in phytopathogen Fusarium oxysporum f.sp. cucumerinum.

Protein O-mannosyltransferases (PMTs) have been identified in fungi but not in plants and nematodes, which makes PMTs become attractive targets for developing a new strategy against phytopathogens. Three PMTs have been identified in Fusarium oxysporum, a fungal pathogen that causes vascular wilt in a broad range of economical crops. By deletion or suppression of the pmt genes, we showed that all mutants displayed retarded growth, reduced conidiation, cell wall defects, ER stress and attenuated virulence in F. oxysporum f.sp. cucumerinum. In addition, the Δpmt1 exhibited reduced thermotolerance, while the Δpmt4 and the pmt2 conditional mutant exhibited abnormal polarized growth. Comparative glycoproteome analysis of these pmt mutants revealed that PMTs preferentially modified random coils with flanking regions rich in Ser, Thr, Ala, Glu, Asp and Lys at the stem region of membrane proteins, the N-terminal region close to signal peptide of secreted proteins, or surface of soluble proteins. PMT1 specifically acted on nuclear proteins and proteins that are responsible for protein folding, which might contribute to thermotolerance. PMT4 specifically acted on the membrane and soluble proteins in secretory pathways, especially the GPI anchoring pathway, which might contribute to synthesis and transportation of GPI anchored proteins and thus polarized growth. PMT2 was responsible for modification of proteins that are required for protein folding and cell wall synthesis, which might make PMT2 essential. Our results gave an insight to understanding of the roles of each O-mannosyltransferase in F. oxysporum f.sp. cucumerinum and provide a new perspective to prevent Fusarium wilt.

MaPmt1, a protein O-mannosyltransferase, contributes to virulence through governing the appressorium turgor pressure in Metarhizium acridum.

O-glycosylation is a very important post-translational modification of protein and involved in many cell processes in fungi. There exist three protein O-manosyltransferanse genes (MaPmt1, MaPmt2, MaPmt4) in Metarhizium acridum based on sequence homology. Here, MaPmt1, a gene for Pmt1 O-manosyltransferanse in M. acridum, was characterized and functionally analyzed through targeted gene disruption and complementation methods. Deletion of MaPmt1 had no effect on conidial germination, but slightly increased the conidial yield and significantly impaired fungal tolerances to UV-B radiation and wet-heat. Deletion of MaPmt1 made the fungus become more sensitive to cell wall disturbing agents and exhibit a thinner cell wall with changed components. Insect bioassays showed that disruption of MaPmt1 attenuated the fungal virulence significantly by topical inoculation but not by injection, indicating that MaPmt1 is required for penetration during the infection of M. acridum. Interestingly, deletion of MaPmt1 did not affect appressorium formation but significantly decreased appressorium turgor pressure. Moreover, the decreased virulence of MaPmt1 disruptant is mainly due to the reduced appressorium turgor pressure, which may be resulted from the declined glycerol concentration, combined with the weakened cell wall that could not hold the normal appressorium turgor pressure to penetrate the host cuticle.

MaPmt4, a protein O-mannosyltransferase, contributes to cell wall integrity, stress tolerance and virulence in Metarhizium acridum.

In eukaryotic cells, protein O-glycosylation is an essential protein modification. Analysis of the Metarhizium acridum genome database revealed a total of three O-glycoside mannosyltransferase homologs (Pmt1, Pmt2 and Pmt4), closely related to Saccharomyces cerevisiae Pmt1, Pmt2, and Pmt4. In this study, the functions of MaPmt4, encoding a protein O-mannosyltransferase in M. acridum, were characterized using disruption and complementation strategies. Disruption of MaPmt4 delayed the conidial germination and reduced the fungal tolerances to heat shock and UV-B irradiation, but did not affect conidial yield. Inactivation of MaPmt4 displayed increased sensitivity to cell wall-perturbing agents, formed thinner cell walls, and changed composition of fungal cell wall, demonstrating that MaPmt4 was also important to maintain fungal cell wall integrity. Bioassays by topical inoculation and intrahemocoel injection showed that the MaPmt4 deletion mutant exhibited greatly reduced virulence. The subsequent examination revealed that the inactivation of MaPmt4 impaired appressorium formation, decreased fungal growth in locust hemolymph in vitro, and boosted insect immune responses, the latter in part potentially owing to the changes in conidial surface structures, and thus attenuated the virulence of MaPmt4 deletion mutant. Furthermore, the results of comparative proteomics showed that MaPmt4 played important roles in fungal cell wall integrity, stress tolerances, and virulence via broad genetic pathways.

Use of the cysteine-repressible HpMET3 promoter as a novel tool to regulate gene expression in Hansenula polymorpha.

OBJECTIVES: The promoter of HpMET3, encoding an ATP sulfurylase, was evaluated for its potential as a repressible promoter to downregulate the expression of target genes in the thermotolerant, methylotrophic yeast Hansenula polymorpha. RESULTS: The expression of lacZ under the control of the 0.6 kb HpMET3 promoter was efficiently downregulated by cysteine, but not by methionine or sulfate. The HpMET3 promoter was used to generate a conditional mutant of the HpPMT2 gene encoding an O-mannosyltransferase, which is involved in post-translational protein modification. The addition of 0.5 mM cysteine adversely affected the growth of the conditional HpMET3(p)-Hppmt2 mutant strain by downregulating transcription of HpPMT2 to approx. 40 % of the normal levels, indicating that the HpPMT2 gene is essential for cell viability. However, the HpMET3 promoter was neither induced nor repressed in the heterologous host Saccharomyces cerevisiae. CONCLUSION: Our results reveal that the cysteine-repressible HpMET3 promoter is a useful tool that downregulates the expression of various genes in H. polymorpha.

Protein O-mannosylation: what we have learned from baker's yeast.

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.

Effect of ATG8 or SAC1 deficiency on the cell proliferation and lifespan of the long-lived PMT1 deficiency yeast cells.

Autophagy is pivotal in maintaining intracellular homeostasis, which involves various biological processes, including cellular senescence and lifespan modulation. Being an important member of the protein O-mannosyltransferase (PMT) family of enzymes, Pmt1p deficiency can significantly extend the replicative lifespan (RLS) of yeast cells through an endoplasmic reticulum (ER) unfolded protein response (UPR) pathway, which is participated in protein homeostasis. Nevertheless, the mechanisms that Pmt1p regulates the lifespan of yeast cells still need to be explored. In this study, we found that the long-lived PMT1 deficiency strain (pmt1Δ) elevated the expression levels of most autophagy-related genes, the expression levels of total GFP-Atg8 fusion protein and free GFP protein compared with wild-type yeast strain (BY4742). Moreover, the long-lived pmt1Δ strain showed the greater dot-signal accumulation from GFP-Atg8 fusion protein in the vacuole lumen through a confocal microscope. However, deficiency of SAC1 or ATG8, two essential components of the autophagy process, decreased the cell proliferation ability of the long-lived pmt1Δ yeast cells, and prevented the lifespan extension. In addition, our findings demonstrated that overexpression of ATG8 had no potential effect on the RLS of the pmt1Δ yeast cells, and the maintained incubation of minimal synthetic medium lacking nitrogen (SD-N medium as starvation-induced autophagy) inhibited the cell proliferation ability of the pmt1Δ yeast cells with the culture time, and blocked the lifespan extension, especially in the SD-N medium cultured for 15 days. Our results suggest that the long-lived pmt1Δ strain enhances the basal autophagy activity, while deficiency of SAC1 or ATG8 decreases the cell proliferation ability and shortens the RLS of the long-lived pmt1Δ yeast cells. Moreover, the maintained starvation-induced autophagy impairs extension of the long-lived pmt1Δ yeast cells, and even leads to the cell death.

O-Mannosyltransferase CfPmt4 Regulates the Growth, Development and Pathogenicity of Colletotrichum fructicola.

Camellia oleifera is a woody, edible-oil plant native to China. Anthracnose is the major disease of Ca. oleifera, and Colletotrichum fructicola is the main epidemic pathogen. Our previous research indicated that CfHac1 (homologous to ATF/CREB1) and CfGcn5 (general control nonderepressible 5, Gcn5) are integral to key cellular processes that govern fungal development and pathogenesis. Further transcriptomic analyses of the CfHac1 and CfGcn5 mutants, particularly under conditions of endoplasmic reticulum (ER) stress, hold the potential to unveil additional genes implicated in this critical cellular response. We identified all OST/PMT (oligosaccharyltransferase/Protein O-Mannosyltransferases) genes in C. fructicola and analyzed their expression levels. To elucidate novel glycosylation-related genes that may be important for the virulence of C. fructicola, we took an unbiased transcriptomic approach comparing wild-type and the ∆Cfhac1 mutant. Notably, all OST/PMT genes were induced by dithiothreitol and down-regulated in the ΔCfhac1 mutant, yet only the CfPMT4 (Protein O-Mannosyltransferases 4) gene (A04626) was unaffected in the ΔCfgcn5. The results of targeted gene deletion experiments indicate that CfPMT4 plays a crucial role in both vegetative growth and conidiation. Additionally, our investigation revealed that the ΔCfpmt4 exhibits deficiencies in appressorium formation, as well as in its response to cell wall integrity and endoplasmic reticulum stresses. Furthermore, the mutant displayed impaired glycogen metabolism, which may contribute to reduced penetration ability. Overall, CfPmt4, an O-mannosyltransferase, controls the growth, development, and pathogenicity of Colletotrichum fructicola. Understanding the function of the CfPMT4 homolog could provide a potential molecular target for controlling Ca. oleifera anthracnose.

Inhibition of glycolysis represses the growth and alleviates the endoplasmic reticulum stress of breast cancer cells by regulating TMTC3.

Considering the role of glycolysis inhibition as a novel therapeutic strategy for cancer, including breast cancer (BC), we wondered whether glycolysis could affect BC progression by regulating transmembrane O-mannosyltransferase-targeting cadherins 3 (TMTC3). Following the intervention, lactic acid production in BC cells was monitored, and viability, proliferation, and apoptosis assays were performed. The expressions of TMTC3 and endoplasmic reticulum (ER) stress- and apoptosis-related factors Caspase-12, C/EBP homologous protein (CHOP), glucose-regulated protein 78 (GRP78), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X (Bax) were quantified. TMTC3 was lowly expressed in BC tissue and cell. The promotion of glycolysis via glucose represses TMTC3 expression and apoptosis yet enhances lactic acid production and growth of BC cell, along with promoted levels of Caspase-12, CHOP, GRP78, and Bcl-2 yet repressed level of Bax, while the contrary results were evidenced after 2-deoxyglycouse intervention. Overexpressed TMTC3 additionally abrogated the effects of glycolysis on increasing the viability and proliferation yet inhibiting the apoptosis of BC cells, with the increased expressions of Caspase-12, CHOP, and GRP78, and Bcl-2 yet decreased level of Bax. Collectively, inhibiting glycolysis restrained the growth and attenuated the ER stress of BC cell by regulating TMTC3.

Protein O-mannosyltransferase Rv1002c contributes to low cell permeability, biofilm formation in vitro, and mycobacterial survival in mice.

Mycobacterium tuberculosis (M. tuberculosis) Rv1002c encodes the protein O-mannosyltransferase (PMT), which catalyzes the transfer of mannose to serine or threonine residues of proteins. We explored the function of PMT in vitro and in vivo. Rv1002c protein was heterogeneously overexpressed in nonpathogenic Mycobacterium smegmatis (named as MS_Rv1002c). A series of trials including mass spectrometry, transmission electron microscope, biofilm formation and antibiotics susceptibility were performed to explore the function of PMT on bacterial survival in vitro. Mouse experiments were carried out to evaluate the virulence of PMT in vivo. PMT decreased the cell envelope permeability and promoted microbial biofilm formation. PMT enhanced the mycobacterial survival in vivo and inhibited the release of pro-inflammatory cytokines in serum. The function might be associated with an increased abundance of some mannoproteins in culture filtrate (CF). PMT is likely to be involved in mycobacterial survival both in vivo and in vitro due to increasing the mannoproteins abundance in CF.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

A homozygous mutation in the POMT2 gene in four siblings with limb-girdle muscular dystrophy 2N.

Mutations in protein O-mannosyltransferase 2 can cause a wide spectrum of clinical phenotypes from severe congenital muscular dystrophy such as Walker-Warburg syndrome to milder limb-girdle muscular dystrophy 2N. We aimed to describe the clinical and paraclinical features, laboratory tests, and molecular findings of four siblings with a homozygous mutation in the protein O-mannosyltransferase 2 gene. There were two sisters and two brothers, aged 4 to 17 years, with an age of onset symptoms at 3 to 12 years. The main neurologic findings were mild intellectual disability, hypoactive deep tendon reflexes, symmetrical weakness of the proximal lower and/or upper limbs, and difficulties in walking on heels and/or toes. The scoliosis found in two siblings has not been associated with protein O-mannosyltransferase 2 gene mutations related to limb-girdle muscular dystrophy 2N in previous reports. This report expands the phenotypic spectrum of protein O-mannosyltransferase 2 gene mutation-related limb-girdle muscular dystrophy 2N.

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.

Novel compound variants of the TMTC3 gene cause cobblestone lissencephaly-like syndrome: A case report.

Biallelic variants in the transmembrane O-mannosyltransferase targeting cadherins 3 (TMTC3) gene have been reported to cause two distinct types of neuron migration defect diseases, known as cobblestone lissencephaly (COB) and periventricular nodular heterotopia (PVNH), combined with intellectual disability and nocturnal seizures. The aim of the current study was to identify the genetic cause of a 22-month-old Chinese boy who presented with white matter plaques, a small frontal lobe, myelin dysplasia, microcephaly, psychomotor delay, language development delay, truncal hypotonia, intractable epilepsy, infantile spasm and bilateral single transverse palmar creases. Whole-exome sequencing revealed novel heterozygous variant compounds in the TMTC3 gene (c.1123G>A, p.Glu375Lys and c.1126_1129del, p.Arg376Tyrfs*13). Most of the clinical features of the patient are consistent with COB. However, the deformities in the brain (white matter plaques, small frontal lobe and myelin dysplasia) in the patient were more severe compared with those generally exhibited by PVNH, but less severe compared with those presented by COB. Moreover, the patient exhibited bilateral single transverse palmar creases, which, to the best of our knowledge, have not been described previously in patients with a TMTC3 variation. In summary, the current study reported a pediatric Chinese patient with COB-like syndrome caused by TMTC3 gene variations. The present results indicated that variation in the TMTC3 gene can lead to highly variable clinical phenotypes.

Characterization of the Streptomyces coelicolor Glycoproteome Reveals Glycoproteins Important for Cell Wall Biogenesis.

The physiological role of protein O-glycosylation in prokaryotes is poorly understood due to our limited knowledge of the extent of their glycoproteomes. In Actinobacteria, defects in protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation have been shown to significantly retard growth (Mycobacterium tuberculosis and Corynebacterium glutamicum) or result in increased sensitivities to cell wall-targeting antibiotics (Streptomyces coelicolor), suggesting that protein O-glycosylation has an important role in cell physiology. Only a single glycoprotein (SCO4142, or PstS) has been identified to date in S. coelicolor Combining biochemical and mass spectrometry-based approaches, we have isolated and characterized the membrane glycoproteome in S. coelicolor A total of ninety-five high-confidence glycopeptides were identified which mapped to thirty-seven new S. coelicolor glycoproteins and a deeper understanding of glycosylation sites in PstS. Glycosylation sites were found to be modified with up to three hexose residues, consistent with what has been observed previously in other ActinobacteriaS. coelicolor glycoproteins have diverse roles and functions, including solute binding, polysaccharide hydrolases, ABC transporters, and cell wall biosynthesis, the latter being of potential relevance to the antibiotic-sensitive phenotype of pmt mutants. Null mutants in genes encoding a putative d-Ala-d-Ala carboxypeptidase (SCO4847) and an l,d-transpeptidase (SCO4934) were hypersensitive to cell wall-targeting antibiotics. Additionally, the sco4847 mutants displayed an increased susceptibility to lysozyme treatment. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation could be affecting enzyme function.IMPORTANCE In prokaryotes, the role of protein glycosylation is poorly understood due to our limited understanding of their glycoproteomes. In some Actinobacteria, defects in protein O-glycosylation have been shown to retard growth and result in hypersensitivity to cell wall-targeting antibiotics, suggesting that this modification is important for maintaining cell wall structure. Here, we have characterized the glycoproteome in Streptomyces coelicolor and shown that glycoproteins have diverse roles, including those related to solute binding, ABC transporters, and cell wall biosynthesis. We have generated mutants encoding two putative cell wall-active glycoproteins and shown them to be hypersensitive to cell wall-targeting antibiotics. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation affects enzyme function.

Aspergillus nidulans Pmts form heterodimers in all pairwise combinations.

Eukaryotic protein O-mannosyltransferases (Pmts) are divided into three subfamilies (Pmt1, Pmt2, and Pmt4) and activity of Pmts in yeasts and animals requires assembly into complexes. In Saccharomyces cerevisiae, Pmt1 and Pmt2 form a heteromeric complex and Pmt 4 forms a homomeric complex. The filamentous fungus Aspergillus nidulans has three Pmts: PmtA (subfamily 2), PmtB (subfamily 1), and PmtC (subfamily 4). In this study we show that A. nidulans Pmts form heteromeric complexes in all possible pairwise combinations and that PmtC forms homomeric complexes. We also show that MsbA, an ortholog of a Pmt4-modified protein, is not modified by PmtC.

Dystroglycanopathy with two novel POMT1 mutations in a Chinese boy with developmental delay and muscular dystrophy.

Alpha-dystroglycanopathies are a group of diseases due to reduced glycosylation of alpha-dystroglycan, which commonly result from mutations in POMT1, POMT2, and POMGnT1. Patients with alpha-dystroglycanopathies present with muscular, cerebral, and ocular involvements with differing severities. We reported a boy who presented with muscular dystrophy, developmental delay, and non-specific white matter lesions. Mutation analysis of POMT1 was performed and revealed two novel mutations, a substitution mutation (c.176T>G) and a duplication mutation (c.2059dupC) which results in premature termination of translation. In-silico prediction in five different platforms concurred that the substitution is damaging, and functional studies by immunofluorescence revealed lack of staining in the carbohydrate moiety of alpha-dystroglycan, confirming the molecular findings in a functional manner. In conclusion, we reported the first case of genetically confirmed alpha-dystroglycanopathy due to mutations in POMT1 in Chinese.