Karen Palter

GAL4 ENHANCER TRAPS EXPRESSED IN THE EMBRYO, LARVAL BRAIN, IMAGINAL DISCS,AND OVARY OF DROSOPHILA

We have screened a collection of approximately 400 GAL4 enhancer trap lines for useful patterns of expression in the embryo, larval brain, imaginal discs, and ovary using a UAS‐lacZ reporter construct. Although similar patterns of expression have previously been reported in the original P[lacZ] enhancer trap screens, these lines are useful for directing ectopic expression of genes in discrete patterns during these stages. In addition, we have identified some unique patterns of expression that have not been previously reported. Dev. Dyn. 209:310–322, 1997.

Genomic structure and sequence analysis of Drosophila melanogaster HSC70 genes

We report the identification of two new members of the Drosophila melanogaster HSP70 gene family, HSC3 and HSC5. DNA sequence analysis predicts that HSC3 encodes a 72-kDa protein with a hydrophobic leader sequence and a C-terminal retrieval tetrapeptide, KDEL, characteristics associated with luminal endoplasmic reticulum (ER) proteins. Sequence analysis predicts that HSC5 encodes a 74-kDa protein with a characteristic mitochondrial leader sequence. We report the deduced amino acid (aa) sequence for the previously identified gene, HSC1. HSC1 encodes a 70-kDa protein lacking a leader sequence and is presumed to have a cytoplasmic localization. A comparison of the deduced aa sequences of these and hsc70 proteins from different species indicates that hsc70 proteins residing in the same intracellular compartment in different organisms are more similar to each other than are hsc70s from the same organism, but different organelles.

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a β-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a β-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the β(1,2)-linked N-acetylglucosamine from the Manα(1,3) branch compared with the Manα(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal β-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing β-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

Expression of a Functional Drosophila melanogaster CMP-sialic Acid Synthetase DIFFERENTIAL LOCALIZATION OF THE DROSOPHILA AND HUMAN ENZYMES

CMP-N-acetylneuraminic acid is a critical metabolite in the generation of glycoconjugates that play a role in development and other physiological processes. Whereas pathways for its generation are firmly established in vertebrates, the presence and function of the relevant synthetic enzyme in insects and other protostomes is unknown. In this study, we characterize the first functional CMP-sialic acid synthase (DmCSAS) from any protostome lineage expressed from a D. melanogaster cDNA clone. Homologous genes were subsequently identified in other insect species. The gene is developmentally regulated, with expression first appearing at 12–24 h of embryogenesis, low expression through larval and pupal stages, and greatly enriched expression in the adult head, suggesting a possible role in the central nervous system. Activity of the enzyme was verified by an increase in in vitro and in vivo CMP-N-acetylneuraminic acid levels when expressed in a heterologous host. Unlike all known vertebrate CMP-sialic acid synthetase (CSAS) proteins that localize to the nucleus, the D. melanogaster CSAS protein was targeted to the Golgi compartment when expressed in both heterologous mammalian and insect cell lines. Replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence resulted in the redirection of the chimeric CSAS protein to the nucleus but with a concomitant loss of enzymatic activity. The localization of CSAS orthologs to different intracellular organelles represents, to our knowledge, the first example of differential protein targeting of orthologs in eukaryotes and reveals how the sialylation pathway diverged during the evolution of protostomes and deuterostomes.

GlycoFly: a database of Drosophila N-linked glycoproteins identified using SPEG--MS techniques.

Protein glycosylation affects cellular functions of the central nervous system (CNS). Its deficiency leads to neurological disorders such as ataxia, paralysis, learning disability, mental retardation, and memory loss. However, the glycoproteins that are responsible for these diseases are not well characterized. In this study, Drosophila melanogaster was used as a model organism to identify the N-glycosylated proteins and N-glycosylation sites of its CNS by means of proteomics. Adult fly heads were digested with chymotrypsin or trypsin and the N-linked glycopeptides were captured using solid phase extraction of N-linked glycopeptides (SPEG) technique followed by mass spectrometry (MS) analysis using LTQ OrbiTrap Velos. Three hundred and thirty new and 147 previously known glycoproteins were identified from 721 uniquely detected peptides that have 740 NXS/T glycosylation sites. The N-glycosylation sites were highly abundant in cell adhesion, ion channel, and ion binding molecules, which are important for nerve maturation, organ development, axon guidance, learning, and memory. Identification of the N-glycosylated sites of these proteins will enhance our knowledge of these proteins and serve as a basis for future studies to address the roles of these proteins in neurological function and disorders. A database for Drosophila N-linked glycopeptides ( http://betenbaugh.jhu.edu/GlycoFly ) has been established in this study as a resource for study of neurological disorders.

GlycoFish: A Database of Zebrafish N-linked Glycoproteins Identified Using SPEG Method Coupled with LC/MS

Zebrafish (Danio rerio) is a model organism that is used to study the mechanisms and pathways of human disorders. Many dysfunctions in neurological, development, and neuromuscular systems are due to glycosylation deficiencies, but the glycoproteins involved in zebrafish embryonic development have not been established. In this study, a mass spectrometry-based glycoproteomic characterization of zebrafish embryos was performed to identify the N-linked glycoproteins and N-linked glycosylation sites. To increase the number of glycopeptides, proteins from zebrafish were digested with two different proteases--chymotrypsin and trypsin--into peptides of different length. The N-glycosylated peptides of zebrafish were then captured by the solid-phase extraction of N-linked glycopeptides (SPEG) method and the peptides were identified with an LTQ OrbiTrap Velos mass spectrometer. From 265 unique glycopeptides, including 269 consensus NXT/S glycosites, we identified 169 different N-glycosylated proteins. The identified glycoproteins were highly abundant in proteins belonging to the transporter, cell adhesion, and ion channel/ion binding categories, which are important to embryonic, organ, and central nervous system development. This proteomics data will expand our knowledge about glycoproteins in zebrafish and may be used to elucidate the role that glycosylation plays in cellular processes and disease. The glycoprotein data are available through the GlycoFish database (http://betenbaugh.jhu.edu/GlycoFish) introduced in this paper.

DmSAS Is Required for Sialic Acid Biosynthesis in Cultured Drosophila Third Instar Larvae CNS neurons

Sialylation is an important carbohydrate modification of glycoconjugates that has been shown to modulate many cellular/molecular interactions in vertebrates. In Drosophila melanogaster (Dm), using sequence homology, several enzymes of the sialylation pathway have been cloned and their function tested in expression systems. Here we investigated whether sialic acid incorporation in cultured Dm central nervous system (CNS) neurons required endogenously expressed Dm sialic acid synthase (DmSAS). We compared neurons derived from wild type Dm larvae with those containing a DmSAS mutation (148 bp deletion). The ability of these cells to produce Sia5NAz (sialic acid form) from Ac(4)ManNAz (azide-derivatized N-acetylmannosamine) and incorporate it into their glycoconjugates was measured by tagging the azide group of Sia5NAz with fluorescent agents via Click-iT chemistry. We found that most of the wild type Dm CNS neurons incorporated Sia5NAz into their glycoconjugates. Sialic acid incorporation was higher at the soma than at the neurite and could also be detected at perinuclear regions and the plasma membrane. In contrast, neurons from the DmSAS mutant did not incorporate Sia5NAz unless DmSAS was reintroduced (rescue mutant). Most of the neurons expressed α2,6-sialyltransferase. These results confirm that the mutation was a null mutation and that no redundant sialic acid biosynthetic activity exists in Dm cells, i.e., there is only one DmSAS. They also provide the strongest proof to date that DmSAS is a key enzyme in the biosynthesis of sialic acids in Dm CNS neurons, and the observed subcellular distribution of the newly synthesized sialic acids offers insights into their biological function.

Measurement of sialic acid content on recombinant membrane proteins

BackgroundMembrane proteins such as cell adhesion molecules,receptors, transporters and ion channel proteins all haveessential roles in cell-growth, migration, and flow ofinformation, cell-cell and cell-protein communication.Membrane proteins are targets of biopharmaceuticalcompanies because they have diverse effects on the pro-gression of many diseases[1]. Ion channels are mem-brane proteins that play critical roles in a number ofcell functions including communication and neuromus-cular activity. Treatment of channelopathy diseases suchas cancer, cardiac arrhythmia, ataxia, paralysis, epilepsy,memory and learning loss, requires a broad understand-ing of ion channel function.Sialic acid is a criticalcharged glycan that affects the action potential of potas-sium channels which leads to changes in the neuronalsystem of organisms. In order to understand the effectof the sialylation on channel function, the presence ofthe sialic acid on the protein of interest should be stu-died. In this study, a novel method for the quantificationof sialic acid is described for a potassium channel mem-brane protein.Materials and methods