A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is essential for viability in Drosophila melanogaster

We report the first demonstration that the activity of a member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family is necessary for viability in Drosophila melanogaster. Expression of the wild-type recombinant pgant35A gene in COS7 cells resulted in in vitro activity against peptide and glycopeptide substrates, demonstrating that this gene encodes a biochemically active transferase. Previous mutagenesis studies identified recessive lethal mutations that were rescued by a genomic fragment containing the pgant35A gene; however, the presence of additional open reading frames within this fragment left open the possibility that another gene was responsible for rescue of the observed lethality. Here, we have determined the molecular nature of the mutations in three independent mutant alleles. Two of the mutant alleles contain premature stop codons within the coding region of pgant35A. The third mutant contains an arginine to tryptophan amino acid change, which, when expressed in COS7 cells, resulted in a dramatic reduction of transferase activity in vitro. PCR amplification of this gene from Drosophila cDNA panels and Northern analysis revealed that it is expressed throughout embryonic, larval, and pupal stages as well as in adult males and females. This study provides the first direct evidence for the involvement of a member of this conserved multigene family in eukaryotic development and viability.     

Rescue of <Emphasis Type="Italic">Drosophila Melanogaster l(2)35Aa</Emphasis> lethality is only mediated by polypeptide GalNAc-transferase <Emphasis Type="Italic">pgant35A</Emphasis>, but not by the evolutionary conserved human ortholog GalNAc-transferase-T11

The Drosophila l(2)35Aa gene encodes a UDP-N-acetylgalactosamine: Polypeptide N-acetylgalactosaminyltransferase, essential for embryogenesis and development (J. Biol. Chem. 277, 22623–22638; J. Biol. Chem. 277, 22616–22). l(2)35Aa, also known as pgant35A, is a member of a large evolutionarily conserved family of genes encoding polypeptide GalNAc-transferases. Phylogenetic and functional analyses have proposed that subfamilies of orthologous GalNAc-transferase genes are conserved in species, suggesting that they serve distinct functions in vivo. Based on sequence alignments, pgant35A and human GALNT11 are thought to belong to a distinct subfamily. Recent in vitro studies have shown that pgant35A and pgant7, encoding enzymes from different subfamilies, prefer different acceptor substrates, whereas the orthologous pgant35A and human GALNT11 gene products possess, 1) conserved substrate preferences and 2) similar acceptor site preferences in vitro. In line with the in vitro pgant7 studies, we show that l(2)35Aa lethality is not rescued by ectopic pgant7 expression. Remarkably and in contrast to this observation, the human pgant35A ortholog, GALNT11, was shown not to support rescue of the l(2)35Aa lethality. By use of genetic “domain swapping” experiments we demonstrate, that lack of rescue was not caused by inappropriate sub-cellular targeting of functionally active GalNAc-T11. Collectively our results show, that fly embryogenesis specifically requires functional pgant35A, and that the presence of this gene product during fly embryogenesis is functionally distinct from other Drosophila GalNAc-transferase isoforms and from the proposed human ortholog GALNT11.     

A mucin-type O-glycosyltransferase modulates cell adhesion during Drosophila development

Cell-cell and cell-matrix adhesion are crucial during many stages of eukaryotic development. Here, we provide the first example that mucin-type O-linked glycosylation is involved in a developmentally regulated cell adhesion event in Drosophila melanogaster. Mutations in one member of the evolutionarily conserved family of enzymes that initiates O-linked glycosylation alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNA interference to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Expression of wild type pgant3 in the mutant background rescued the wing blistering phenotype, whereas expression of another family member (pgant35A) did not, revealing a unique requirement for pgant3. pgant3 mutants displayed reduced O-glycosylation along the basal surface of larval wing imaginal discs, which was restored with wild type pgant3 expression, suggesting that reduced glycosylation of basal proteins is responsible for disruption of adhesion in the adult wing blade. Glycosylation reactions demonstrated that PGANT3 glycosylates certain extracellular matrix (ECM) proteins. Immunoprecipitation experiments revealed that PGANT3 glycosylates tiggrin, an ECM protein known to bind integrin. We propose that this glycosyltransferase is uniquely responsible for glycosylating tiggrin in the wing disc, thus modulating proper cell adhesion through integrin-ECM interactions. This study provides the first evidence for the role of O-glycosylation in a developmentally regulated, integrin-mediated, cell adhesion event and reveals a novel player in wing blade formation during Drosophila development.     

Multiple members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila

Mucin-type O-glycosylation represents a major form of post-translational modification that is conserved across most eukaryotic species. This type of glycosylation is initiated by a family of enzymes (GalNAc-Ts in mammals and PGANTs in Drosophila) whose members are expressed in distinct spatial and temporal patterns during development. Previous work from our group demonstrated that one member of this family is essential for viability and another member modulates extracellular matrix composition and integrin-mediated cell adhesion during development. To investigate whether other members of this family are essential, we employed RNA interference (RNAi) to each gene in vivo. Using this approach, we identified 4 additional pgant genes that are required for viability. Ubiquitous RNAi to pgant4, pgant5, pgant7, or the putative glycosyltransferase CG30463 resulted in lethality. Tissue-specific RNAi was also used to define the specific organ systems and tissues in which each essential family member is required. Interestingly, each essential pgant had a unique complement of tissues in which it was required. Additionally, certain tissues (mesoderm, digestive system, and tracheal system) required more than one pgant, suggesting unique functions for specific enzymes in these tissues. Expanding upon our RNAi results, we found that conventional mutations in pgant5 resulted in lethality and specific defects in specialized cells of the digestive tract, resulting in loss of proper digestive system acidification. In summary, our results highlight essential roles for O-glycosylation and specific members of the pgant family in many aspects of development and organogenesis.     

Conservation of peptide acceptor preferences between Drosophila and mammalian polypeptide-GalNAc transferase ortholog pairs

UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltrans- ferases (ppGalNAc Ts) comprise a large family of glycosyltransferases that initiate mucin-type protein O-glycosylation, transferring alpha-GalNAc to Thr and Ser residues of polypeptide acceptors. Families of ppGalNAc Ts are found across diverse eukaryotes with orthologs identifiable from mammals to single-cell organisms. The peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly understood. Previously, we reported a series of oriented random peptide substrate libraries for quantitatively determining the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 (Gerken TA, Raman J, Fritz TA, Jamison O. 2006. Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 & T2 (ppGalNAc T1 & T2) derived from oriented random peptide substrates. J Biol Chem. 281:32403-32416). With these substrates, previously unknown features of the transferases were revealed. Utilizing these and a new lengthened set of random peptides, studies have now been performed on PGANT5 and PGANT2, the Drosophila orthologs of T1 and T2. The results from these studies suggest that the major peptide substrate determinants for these transferases are contained within 2 to 3 residues flanking the site of glycosylation. It is further found that the mammalian and fly T1 orthologs display very similar peptide substrate preferences, while the T2 orthologs are nearly indistinguishable, suggesting similar peptide preferences amongst orthologous pairs have been maintained across evolution. This conclusion is further supported by sequence homology comparisons of each of the transferase orthologs, showing that the peptide substrate and UDP binding site residues are more highly conserved between species relative to their remaining catalytic and lectin domain residues.     

Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 and T2 derived from oriented random peptide substrates

A large family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAc Ts) catalyzes the first step of mucin-type protein O-glycosylation by transferring GalNAc to serine and threonine residues of acceptor polypeptides. The acceptor peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly characterized and poorly understood, despite the fact that mutations in two individual isoforms are deleterious to man and the fly. In this work a series of oriented random peptide substrate libraries, based on the GAGAXXXTXXXAGAGK sequence motif (where X = randomized positions), have been used to obtain the first comprehensive determination of the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 isoforms. ppGalNAc T-glycosylated random peptides were isolated by lectin affinity chromatography, and transferase amino acid preferences were determined by Edman amino acid sequencing. The results reveal common and unique position-sensitive features for both transferases, consistent with previous reports of the preferences of ppGalNAc T1 and T2. The random peptide substrates also reveal additional specific features that have never been described before that are consistent with the x-ray crystal structures of the two transferases and furthermore are reflected in a data base analysis of in vivo O-glycosylation sites. By using the transferase-specific preferences, optimum and selective acceptor peptide substrates have been generated for each transferase. This approach represents a relatively complete, facile, and reproducible method for obtaining ppGalNAc T peptide substrate specificity. Such information will be invaluable for identifying isoform-specific peptide acceptors, creating isoform-specific substrates, and predicting O-glycosylation sites.     

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is required for epithelial tube formation

Epithelial tubes are essential for the proper function of a diverse array of eukaryotic organs. Here we present a novel class of genes required for maintaining epithelial cell shape, polarity, and paracellular barrier function in the Drosophila embryonic tracheal system. Mutations in one member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family (pgant35A) are recessive lethal and result in tracheal tubes that are irregular in diameter and morphology. Further analysis of the pgant35A mutants reveals diminished levels of the apical determinant Crbs and the luminal marker 2A12, concomitant with increased staining in cytoplasmic vesicles within tracheal cells. GalNAc-containing glycoproteins are severely diminished along the apical region of the tracheal system as well. Tracheal cells become irregular in size and shape, and septate junction proteins are mislocalized to a more apical position. Most notably, paracellular barrier function is lost in the tracheal system of the mutants. Overexpression of wild type pgant35A under control of the trachea-specific breathless (btl) promoter results in partial rescue of the lethality. We propose a model where pgant35A is required to establish proper apical composition of tracheal cells by influencing apical delivery of proteins/glycoproteins. Disruption of the normal apical content results in altered cell morphology and loss of paracellular barrier function. These studies demonstrate a previously unrecognized requirement for mucin-type O-glycosylation in epithelial tube integrity and have obvious implications for epithelial morphogenesis in higher eukaryotes, since a unique ortholog to pgant35A exists in mammals.     

Identification and expression analysis of Drosophila melanogaster genes encoding beta-hexosaminidases of the sperm plasma membrane

Sperm surface beta-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two beta-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for beta-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the alpha-subunit of the mammalian lysosomal beta-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the beta-subunit of all known beta-N-acetylhexosaminidases, which we have named beta(1) and beta(2), respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an alphabeta(2) structure, and DmHEXB, with a beta(1)beta(2) structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding beta-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional beta-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.     

A calcium-inhibited Drosophila adenylyl cyclase

Mammals possess a family of transmembrane, G-protein-responsive adenylyl cyclase isoforms (tmACs) encoded by distinct genes differing in their patterns of expression and modes of biochemical regulation. Our previous work confirmed that Drosophila melanogaster also possesses a family of tmAC isoforms defining the fly as a suitable genetic model for discerning mammalian tmAC function. We now describe a Drosophila tmAC, DAC39E, which employs a novel means for regulating its expression; differential exon utilization results in a developmental switch in DAC39E protein. DAC39E protein sequence is most closely related to mammalian type III AC, and it is predominantly expressed in the central nervous system (CNS) and olfactory organs, suggesting a role in processing sensory signaling inputs. DAC39E catalytic activity is inhibited by micromolar concentrations of calcium; therefore, DAC39E is oppositely regulated by calcium compared to the only other tmAC shown to be expressed in the Drosophila CNS, Rutabaga AC. The presence of both positively and negatively regulated tmACs suggests a complex mode of cross-talk between cAMP and calcium signal transduction pathways in the fly CNS.     

Tissue and stage-specific expression of the Tolls in Drosophila embryos

The Drosophila transmembrane receptor Toll plays a key role in specifying the dorsoventral axis of the embryo. At later stages of development, it controls the immune response of the fly to fungal and Gram-positive bacterial infections. The Drosophila genome has a total of nine Toll-like genes, including the previously characterized Toll (Toll-1) and 18-wheeler (Toll-2). Here we describe the embryonic expression patterns of the seven Toll-like genes Toll-3 through Toll-9. We find that these genes have distinct expression domains and that their expression is dynamically changing throughout embryonic development. This complex and tissue-specific regulation of Toll-like gene expression strongly suggests a role in embryonic development for most Drosophila Tolls. The evolving picture on the Toll family members in Drosophila contrasts with that of mammalian Toll-like receptors, which are predominantly expressed in immune responsive cells where their activation occurs via microbial structural determinants.     

Characterization of mammalian orthologues of the Drosophila osa gene: cDNA cloning, expression, chromosomal localization, and direct physical interaction with Brahma chromatin-remodeling complex

The osa gene of Drosophila melanogaster encodes a nuclear protein that is a component of the Brahma chromatin-remodeling complex. Osa is required for embryonic segmentation, development of the notum and wing margin, and photoreceptor differentiation. In these tissues, osa mutations have effects opposite to those caused by wingless (wg) mutations, suggesting that osa functions as an antagonist of wg signaling. Here we describe the cloning and characterization of mammalian orthologues of osa. Three evolutionarily conserved domains were identified in Osa family members: the N-terminal Bright domain and C-terminally located Osa homology domains 1 and 2. RNase protection analysis indicates a widespread expression of the Osa1 gene during mouse development, in adult tissues, and in cultured cell lines. The Osa1 gene was localized to mouse chromosome 4, within the region syntenic to chromosomal position 1p35-p36 of its human counterpart. We present evidence that the OSA1 product is localized in the nucleus and associates with human Brahma complex, which suggests evolutionarily conserved function for Osa in gene regulation between mammals and Drosophila.     

A Dnmt2-like protein mediates DNA methylation in Drosophila

The methylation status of Drosophila DNA has been discussed controversially over a long time. Recent evidence has provided strong support for the existence of 5-methylcytosine in DNA preparations from embryonic stages of fly development. The Drosophila genome contains a single candidate DNA methyltransferase gene that has been termed Dnmt2. This gene belongs to a widely conserved family of putative DNA methyltransferases. However, no catalytic activity has been demonstrated for any Dnmt2-like protein yet. We have now established a protocol for the immunological detection of methylated cytosine in fly embryos. Confocal analysis of immunostained embryos provided direct evidence for the methylation of embryonic DNA. In order to analyse the function of Dnmt2 in DNA methylation, we depleted the protein by RNA interference. Depletion of Dnmt2 had no detectable effect on embryonic development and resulted in a complete loss of DNA methylation. Consistently, overexpression of Dnmt2 from an inducible transgene resulted in significant genomic hypermethylation at CpT and CpA dinucleotides. These results demonstrate that Dnmt2 is both necessary and sufficient for DNA methylation in Drosophila and suggest a novel CpT/A-specific DNA methyltransferase activity for Dnmt2 proteins.     

Hits, Fhits and Nits: beyond enzymatic function

We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion genewas discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5'-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5'-end of the fly Fhit gene, this gene was hailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have turned out to be at least as mysterious as the Fhit proteins, with the Nit1 substrate still unknown and the surprising finding that Nit proteins also appear to behave as tumor suppressor proteins. Whether the predicted enzymatic functions of these proteins are relevant to the observed biological functions, remain among the outstanding unanswered puzzles and raise the question: have these mammalian proteins evolved beyond the putative original enzymatic purpose, such that the catalytic function is now vestigial and subservient to signal pathways that use the protein-substrate complexes in pathways that signal apoptosis or DNA damage response? Or can these proteins be fulfilling catalytic functions independently but in parallel with signal pathway functions, as perhaps observed for Aprataxin? Or is the catalytic function indeed part of the observed biological functions, such as apoptosis and tumor suppression? Perhaps the recent, post-genomic focus on metabolomics and genome-wide investigations of signal pathway networks will lead to answers to some of these outstanding questions.     

The Drosophila VEGF receptor homolog is expressed in hemocytes.

Several signalling pathways have been defined by studies of genes originally characterised in Drosophila. However, some mammalian signalling systems have so far escaped discovery in the fly. Here, we describe the identification and characterisation of fly homologs for the mammalian vascular endothelial growth factor/platelet derived growth factor (VEGF/PDGF) and the VEGF receptor. The Drosophila factor (DmVEGF-1) gene has two splice variants and is expressed during all stages, the signal distribution during embryogenesis being ubiquitous. The receptor (DmVEGFR) gene has several splice variants; the variations affecting only the extracellular domain. The most prominent form is expressed in cells of the embryonic haematopoietic cell lineage, starting in the mesodermal area of the head around stage 10 of embryogenesis. Expression persists in hemocytes as embryonic development proceeds and the cells migrate posteriorly. In a fly strain carrying a deletion uncovering the DmVEGFR gene, hemocytes are still present, but their migration is hampered and the hemocytes remain mainly in the anterior end close to their origin. These data suggest that the VEGF/PDGF signalling system may regulate the migration of the Drosophila embryonic haemocyte precursor cells.     

Regulation and function of the RSK family of protein kinases

The RSK (90 kDa ribosomal S6 kinase) family comprises a group of highly related serine/threonine kinases that regulate diverse cellular processes, including cell growth, proliferation, survival and motility. This family includes four vertebrate isoforms (RSK1, RSK2, RSK3 and RSK4), and single family member orthologues are also present in Drosophila and Caenorhabditis elegans. The RSK isoforms are downstream effectors of the Ras/ERK (extracellular-signal-regulated kinase) signalling pathway. Significant advances in the field of RSK signalling have occurred in the past few years, including several new functions ascribed to the RSK isoforms, the discovery of novel protein substrates and the implication of different RSK isoforms in cancer. Collectively, these new findings increase the diversity of biological functions regulated by RSK, and highlight potential new directions of research. In the present paper, we review the structure, expression and activation mechanisms of the RSK isoforms, and discuss their physiological roles on the basis of established substrates and recent discoveries.     

Exportin-5 orthologues are functionally divergent among species

Exportin-5, an evolutionarily conserved nuclear export factor belonging to the importin-β family of proteins, is known to play a role in the nuclear export of small noncoding RNAs such as precursors of microRNA, viral minihelix RNA and a subset of tRNAs in mammalian cells. In this study, we show that the exportin-5 orthologues from different species such as human, fruit fly and yeast exhibit diverged functions. We found that Msn5p, a yeast exportin-5 orthologue, binds double-stranded RNAs and that it prefers a shorter 22 nt, double-stranded RNA to ~80 nt pre-miRNA, even though both of these RNAs share a similar terminal structure. Furthermore, we found that Drosophila exportin-5 binds pre-miRNAs and that amongst the exportin-5 orthologues tested, it shows the highest affinity for tRNAs. The knockdown of Drosophila exportin-5 in cultured cells decreased the amounts of tRNA as well as miRNA, whereas the knock down of human exportin-5 in cultured cells affected only miRNA but not tRNA levels. These results indicate that double-stranded RNA binding ability is an inherited functional characteristic of the exportin-5 orthologues and that Drosophila exportin-5 functions as an exporter of tRNAs as well as pre-miRNAs in the fruit fly that lacks the orthologous gene for exportin-t.     

Dissecting the Biological Role of Mucin-type O-Glycosylation Using RNA Interference in Drosophila Cell Culture

Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.