Dataset Information

Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted.

ABSTRACT: Recent studies highlighted an emerging possibility of using Drosophila as a model system for investigating the mechanisms of human congenital muscular dystrophies, called dystroglycanopathies, resulting from the abnormal glycosylation of alpha-dystroglycan. Several of these diseases are associated with defects in O-mannosylation, one of the most prominent types of alpha-dystroglycan glycosylation mediated by two protein O-mannosyltransferases. Drosophila appears to possess homologs of all essential components of the mammalian dystroglycan-mediated pathway; however, the glycosylation of Drosophila Dystroglycan (DG) has not yet been explored. In this study, we characterized the glycosylation of Drosophila DG using a combination of glycosidase treatments, lectin blots, trypsin digestion, and mass spectrometry analyses. Our results demonstrated that DG extracellular domain is O-mannosylated in vivo. We found that the concurrent in vivo activity of the two Drosophila protein O-mannosyltransferases, Rotated Abdomen and Twisted, is required for O-mannosylation of DG. While our experiments unambiguously determined some O-mannose sites far outside of the mucin-type domain of DG, they also provided evidence that DG bears a significant amount of O-mannosylation within its central region including the mucin-type domain, and that O-mannose can compete with O-GalNAc glycosylation of DG. We found that Rotated Abdomen and Twisted could potentiate in vivo the dominant-negative effect of DG extracellular domain expression on crossvein development, which suggests that O-mannosylation can modulate the ligand-binding activity of DG. Taken together these results demonstrated that O-mannosylation of Dystroglycan is an evolutionarily ancient mechanism conserved between Drosophila and humans, suggesting that Drosophila can be a suitable model system for studying molecular and genetic mechanisms underlying human dystroglycanopathies.

SUBMITTER: Nakamura N

PROVIDER: S-EPMC2912551 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted.

Nakamura Naosuke N Stalnaker Stephanie H SH Lyalin Dmitry D Lavrova Olga O Wells Lance L Panin Vladsilav M VM

Glycobiology 20091207 3

Recent studies highlighted an emerging possibility of using Drosophila as a model system for investigating the mechanisms of human congenital muscular dystrophies, called dystroglycanopathies, resulting from the abnormal glycosylation of alpha-dystroglycan. Several of these diseases are associated with defects in O-mannosylation, one of the most prominent types of alpha-dystroglycan glycosylation mediated by two protein O-mannosyltransferases. Drosophila appears to possess homologs of all essent ...[more]

PMID: 19969597

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Project description:The family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2, which are thought to be essential for muscle and neural development. Similar to mammalian organisms, Drosophila has two O-mannosyltransferase genes, rotated abdomen (rt) and DmPOMT2, encoding proteins with high homology to their mammalian counterparts. The previously reported mutant phenotype of the rt gene includes a clockwise rotation of the abdomen and defects in embryonic muscle development. No mutants have been described so far for the DmPOMT2 locus. In this study, we determined that the mutation in the twisted (tw) locus, tw(1), corresponds to a DmPOMT2 mutant. The twisted alleles represent a complementation group of recessive mutations that, similar to the rt mutants, exhibit a clockwise abdomen rotation phenotype. Several tw alleles were isolated in the past; however, none of them was molecularly characterized. We used an expression rescue approach to confirm that tw locus represents DmPOMT2 gene. We found that the tw1 allele represents an amino acid substitution within the conserved PMT domain of DmPOMT2 (TW) protein. Immunostaining experiments revealed that the protein products of both rt and tw genes colocalize within Drosophila cells where they reside in the ER subcellular compartment. In situ hybridization analysis showed that both genes have essentially overlapping patterns of expression throughout most of embryogenesis (stages 8-17), while only the rt transcript is present at early embryonic stages (5 and 6), suggesting its maternal origin. Finally, we analyzed the genetic interactions between rt and tw using several mutant alleles, RNAi, and ectopic expression approaches. Our data suggest that the two Drosophila O-mannosyltransferase genes, rt and tw, have nonredundant functions within the same developmental cascade and that their activities are required simultaneously for possibly the same biochemical process. Our results establish the possibility of using Drosophila as a model system for studying molecular and genetic mechanisms of protein O-mannosylation during development.

Project description: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.

Project description:Practice of movement in virtual-reality and other artificially altered environments has been proposed as a method for rehabilitation following neurological injury and for training new skills in healthy humans. For such training to be useful, there must be transfer of learning from the artificial environment to the performance of desired skills in the natural environment. Therefore an important assumption of such methods is that practice in the altered environment engages the same learning and plasticity mechanisms that are required for skill performance in the natural environment. We test the hypothesis that transfer of learning may fail because the learning and plasticity mechanism that adapts to the altered environment is different from the learning mechanism required for improvement of motor skill. In this paper, we propose that a model that separates skill learning and environmental adaptation is necessary to explain the learning and aftereffects that are observed in virtual reality experiments. In particular, we studied the condition where practice in the altered environment should lead to correct skill performance in the original environment. Our 2-mechanism model predicts that aftereffects will still be observed when returning to the original environment, indicating a lack of skill transfer from the artificial environment to the original environment. To illustrate the model prediction, we tested 10 healthy participants on the interaction between a simple overlearned motor skill (straight hand movements to targets in different directions) and an artificially altered visuomotor environment (rotation of visual feedback of the results of movement). As predicted by the models, participants show adaptation to the altered environment and after-effects on return to the baseline environment even when practice in the altered environment should have led to correct skill performance. The presence of aftereffect under all conditions that involved changes in environment demonstrates separation of environmental adaptation and skill learning. Our results support the existence of two distinct learning modules with different adaptation properties. Therefore we suggest that adaptation to an altered environment may not be useful for training new skills.

Dataset Information

Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted.

Publications

Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted.

Similar Datasets

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