The twisted Gene Encodes Drosophila Protein O-Mannosyltransferase 2 and Genetically Interacts With the rotated abdomen Gene Encoding Drosophila Protein O-Mannosyltransferase 1

Abstract

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 tw 1 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.

The Diversity of O-Linked Glycans Expressed during Drosophila melanogaster Development Reflects Stage- and Tissue-specific Requirements for Cell Signaling

“…The addition of a novel, less prevalent, branched form of the glucuronylated core 1 glycan (Structure 5) increases this portfolio to 98% of the total profile. We also detected a group of expected, minor structures (O-Man, O-Glc, monoxylosylated O-Glc, O-GlcNAc) that have been predicted to exist in embryonic tissues based on phenotypes associated with mutations in the proposed biosynthetic enzymes or on the in vitro identification of appropriate enzymatic activities: protein O-mannosyltransferases, rotated/POMT1 and twisted/POMT2; protein O-glucosyltranferase, rumi/Poglut; and O-GlcNAc transferase (OGT) (8,58,59,(67)(68)(69)(70)(71).…”

Dystroglycan and ProteinO-Mannosyltransferases 1 and 2 Are Required to Maintain Integrity ofDrosophilaLarval Muscles

“…rt 2 and rt p are semilethal P-element insertion alleles as described by Martin-Blanco and Garcia-Bellido (1996). tw 1 is viable and carries an amino acid substitution in the conserved POM domain (Lyalin et al, 2006). Lethal and semilethal alleles were balanced over green fluorescent (GFP)-marked balancers; absence of GFP was used to select homozygous mutant larvae.…”

“…Dg isoforms that lack the mucin-like domain are required to maintain polarity in the follicular epithelium (Schneider et al, 2006), suggesting that the Dg isoforms can have different functional roles in Drosophila. POMTs are also found in Drosophila (Martin-Blanco and Garcia-Bellido, 1996;Ichimiya et al, 2004;Lyalin et al, 2006) and biochemical analysis of Drosophila POMT1, encoded by the gene rotated abdomen (rt; Martin-Blanco and Garcia-Bellido, 1996) and POMT2, encoded by twisted (tw; Lyalin et al, 2006) has demonstrate that these enzymes possess POMT activity similar to their vertebrate homologues (Ichimiya et al, 2004). When transfected together in insect cells they are able to transfer mannose to a GST-␣-Dg substrate generating Olinked mannose (Ichimiya et al, 2004).…”

“…In addition, reducing the level of either enzyme in vivo by RNA interference (RNAi) resulted in reduced POMT activity of larval extracts toward the Dg substrate (Ichimiya et al, 2004). Mutation of the Drosophila enzymes or reducing their function using RNAi results in rotation of adult abdominal muscles, suggesting the enzymes have some role in muscle function or development (Martin-Blanco and Garcia-Bellido, 1996;Ichimiya et al, 2004;Lyalin et al, 2006). However, it is unclear if these phenotypes are related to a role for the enzymes in Dg modification and function because such a phenotype has not been described in Dg mutants.…”

Biosynthetic Pathway of O-Mannosyl Glycan in Mammals

“…O-Mannosylation is essential for maintaining cell shape and cell integrity (21) and contributes to quality control of proteins in the endoplasmic reticulum (ER) in yeast (22,23). Reduction of protein O-mannosyltransferases in Drosophila melanogaster resulted in defects in embryonic muscle development (24)(25)(26)(27) and pomt1 deletion in mouse resulted in embryonic death (28). Recently, we also demonstrated that O-mannosyltranserase was required for muscle development in zebrafish (29).…”