Inflammation-induced transcriptional regulation of Golgi transporters required for the synthesis of sulfo sLex glycan epitopes.

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3'-phosphate 5'-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.

APECED-causing mutations in AIRE reveal the functional domains of the protein.

A defective form of the AIRE protein causes autoimmune destruction of target organs by disturbing the immunological tolerance of patients with a rare monogenic disease, autoimmune polyendocrinopathy (APE)-candidiasis (C)-ectodermal dystrophy (ED), APECED. Recently, experiments on knockout mice revealed that AIRE controls autoimmunity by regulating the transcription of peripheral tissue-restricted antigens in thymic medullary epithelial cells. Thus, AIRE provides a unique model for molecular studies of organ-specific autoimmunity. In order to analyze the molecular and cellular consequences of 16 disease-causing mutations in vitro, we studied the subcellular localization, transactivation capacity, homomultimerization, and complex formation of several mutant AIRE polypeptides. Most of the mutations altered the nucleus-cytoplasm distribution of AIRE and disturbed its association with nuclear dots and cytoplasmic filaments. While the PHD zinc fingers were necessary for the transactivation capacity of AIRE, other regions of AIRE also modulated this function. Consequently, most of the mutations decreased transactivation. The HSR domain was responsible for the homomultimerization activity of AIRE; all the missense mutations of the HSR and the SAND domains decreased this activity, but those in other domains did not. The AIRE protein was present in soluble high-molecular-weight complexes. Mutations in the HSR domain and deletion of PHD zinc fingers disturbed the formation of these complexes. In conclusion, we propose an in vitro model in which AIRE transactivates transcription through heteromeric molecular interactions that are regulated by homomultimerization and conditional localization of AIRE in the nucleus or in the cytoplasm.

The Drosophila hugin gene codes for myostimulatory and ecdysis-modifying neuropeptides.

In a genomic screen we isolated the Drosophila gene hugin (hug, cytology 87C1-2) by cross-hybridisation to a human glial cell line-derived neurotrophic factor cDNA. Upon cDNA sequence analysis and in vitro expression assays, the hugin gene was found to encode a signal peptide containing proprotein that was further processed in Schneider-2 cells into peptides similar to known neuropeptides. Two of the peptides were similar to FXPRL-amides (pyrokinins) and to the ecdysis-triggering hormone, respectively. The former displayed myostimulatory activity in a bioassay on the cockroach hyperneural muscle preparation, as well as in the Drosophila heart muscle assay. Hugin is expressed during the later half of embryogenesis and during larval stages in a subgroup of neurosecretory cells of the suboesophageal ganglion. Ubiquitous ectopic hugin expression resulted in larval death predominantly at or shortly after ecdysis from second to third instar, suggesting that at least one of the posttranslational cleavage products affects molting of the larva by interfering with the regulation of ecdysis.

Composition of Drosophila melanogaster proteome involved in fucosylated glycan metabolism.

The whole genome approach enables the characterization of all components of any given biological pathway. Moreover, it can help to uncover all the metabolic routes for any molecule. Here we have used the genome of Drosophila melanogaster to search for enzymes involved in the metabolism of fucosylated glycans. Our results suggest that in the fruit fly GDP-fucose, the donor for fucosyltransferase reactions, is formed exclusively via the de novo pathway from GDP-mannose through enzymatic reactions catalyzed by GDP-D-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (GMER, also known as FX in man). The Drosophila genome does not have orthologs for the salvage pathway enzymes, i.e. fucokinase and GDP-fucose pyrophosphorylase synthesizing GDP-fucose from fucose. In addition we identified two novel fucosyltransferases predicted to catalyze alpha1,3- and alpha1,6-specific linkages to the GlcNAc residues on glycans. No genes with the capacity to encode alpha1,2-specific fucosyltransferases were found. We also identified two novel genes coding for O-fucosyltransferases and a gene responsible for a fucosidase enzyme in the Drosophila genome. Finally, using the Drosophila CG4435 gene, we identified two novel human genes putatively coding for fucosyltransferases. This work can serve as a basis for further whole-genome approaches in mapping all possible glycosylation pathways and as a basic analysis leading to subsequent experimental studies to verify the predictions made in this work.