A new superfamily of protein-O-fucosyltransferases,alpha2-fucosyltransferases,and alpha6-fucosyltransferases: phylogeny and identification of conserved peptide motifs

The presence of three conserved peptide motifs shared by alpha2-fucosyltransferases, alpha6-fucosyltransferases, the protein-O-fucosyltransferase family 1 (POFUT1) and a newly identified protein-O-fucosyltransferase family 2 (POFUT2), together with evidence that the present genes encoding for these enzymes have originated from a common ancestor by duplication and divergent evolution, suggests that they constitute a new superfamily of fucosyltransferases.     

The serine/threonine kinase Cmk2 is required for oxidative stress response in fission yeast

Cmk2, a fission yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, is essential for oxidative stress response. Cells lacking cmk2 gene were specifically sensitive to oxidative stress conditions. Upon stress, Cmk2 was phosphorylated in vivo, and this phosphorylation was dependent on the stress-activated MAPK Sty1/Spc1. Co-precipitation assays demonstrated that Cmk2 binds Sty1. Furthermore, in vivo or in vitro activated Sty1 was able to phosphorylate Cmk2, and the phosphorylation occurred at the C-terminal regulatory domain at Thr-411. Cell lethality caused by overexpression of Wis1 MAPK kinase was abolished by deletion of cmk2 or by mutation of Thr-411 of Cmk2. Taken together, our data suggest that Cmk2 acts downstream of Sty1 and is an essential kinase for oxidative stress responses.     

Congenital disorders of glycosylation type Ig is defined by a deficiency in dolichyl-P-mannose:Man7GlcNAc2-PP-dolichyl mannosyltransferase

Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for protein N-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast gene ALG12 that encodes the dolichyl-P-Man:Man(7)GlcNAc(2)-PP-dolichyl alpha6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man(7)GlcNAc(2) from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum.     

Production of recombinant C5a from rainbow trout (Oncorhynchus mykiss): role in leucocyte chemotaxis and respiratory burst

Activation of the complement system can lead to the formation of the membrane attack complex, in which the component C5 is cleaved into C5a and C5b fragments. The C5a anaphylatoxin is a very potent pro-inflammatory molecule that induces chemotaxis and respiratory burst processes in a variety of mammalian leucocytes. While C5a has been well studied in mammals, little is known about the structure and function of C5a in teleost fish or other non-mammalian species. In the present study, we have produced and purified recombinant rainbow trout C5a (rtC5a), and we have shown that it plays an important role in inducing leucocyte migration as well as in triggering the respiratory burst of peripheral blood (PBLs) and head kidney leucocytes (HKLs). When the carboxy-terminal Arg was removed from rtC5a, its ability to induce cell migration and superoxide production remained intact. Interestingly, we show that leucocytes migrating towards rtC5a attached to the plate with a well-spread circular morphology, whereas those migrating towards activated trout serum displayed more irregular and dendritic-like shapes. Our data suggest that the basic mechanisms of action of the C5a anaphylotoxin have remained conserved for more than 300 million years.     

Biochemical and functional characterization of the Ebola virus VP24 protein: implications for a role in virus assembly and budding

The VP24 protein of Ebola virus is believed to be a secondary matrix protein and minor component of virions. In contrast, the VP40 protein of Ebola virus is the primary matrix protein and the most abundant virion component. The structure and function of VP40 have been well characterized; however, virtually nothing is known regarding the structure and function of VP24. Wild-type and mutant forms of VP24 were expressed in mammalian cells to gain a better understanding of the biochemical and functional nature of this viral protein. Results from these experiments demonstrated that (i) VP24 localizes to the plasma membrane and perinuclear region in both transfected and Ebola virus-infected cells, (ii) VP24 associates strongly with lipid membranes, (iii) VP24 does not contain N-linked sugars when expressed alone in mammalian cells, (iv) VP24 can oligomerize when expressed alone in mammalian cells, (v) progressive deletions at the N terminus of VP24 resulted in a decrease in oligomer formation and a concomitant increase in the formation of high-molecular-weight aggregates, and (vi) VP24 was present in trypsin-resistant virus like particles released into the media covering VP24-transfected cells. These data indicate that VP24 possesses structural features commonly associated with viral matrix proteins and that VP24 may have a role in virus assembly and budding.     

Cytogenetics, conserved synteny and evolution of chicken fucosyltransferase genes compared to human

Fucosyltransferases appeared early in evolution, since they are present from bacteria to primates and the genes are well conserved. The aim of this work was to study these genes in the bird group, which is particularly attractive for the comprehension of the evolution of the vertebrate genome. Twelve fucosyltransferase genes have been identified in man. The orthologues of theses genes were looked for in the chicken genome and cytogenetically localized by FISH. Three families of fucosyltransferases: alpha6-fucosyltransferases, alpha3/4-fucosyltransferases, and protein-O-fucosyltransferases, were identified in the chicken with their associated genes. The alpha2-fucosyltransferase family, although present in some invertebrates and amphibians was not found in birds. This absence, also observed in Drosophila, may correspond to a loss of these genes by negative selection. Of the eight chicken genes assigned, six fell on chromosome segments where conservation of synteny between human and chicken was already described. For the two remaining loci, FUT9 and FUT3/5/6, the location may correspond to a new small syntenic area or to an insertion. FUT4 and FUT3/5/6 were found on the same chicken chromosome. These results suggest a duplication of an ancestral gene, initially present on the same chromosome before separation during evolution. By extension, the results are in favour of a common ancestor for the alpha3-fucosyltransferase and the alpha4-fucosyltransferase activities. These observations suggest a general mechanism for the evolution of fucosyltransferase genes in vertebrates by duplication followed by divergent evolution.     

Ancestral exonic organization of FUT8, the gene encoding the alpha6-fucosyltransferase, reveals successive peptide domains which suggest a particular three-dimensional core structure for the alpha6-fucosyltransferase family

Based on PCR strategies and expression studies, we define the genomic organization of the FUT8b gene. This gene encodes the only known mammalian enzyme transferring fucose in an alpha1-->6 linkage on the asparagine-branched GlcNAc residue of the chitobiose unit of complex N:-glycans. The intron/exon organization of the bovine coding sequence determines five successive functional domains. The first exon encodes a domain homologous to cytoskeleton proteins, the second presents a proline-rich region including a motif XPXPPYXP similar to the peptide ligand of the SH3-domain proteins, the third encodes a gyrase-like domain (an enzyme which can bind nucleotides), and the fourth encodes a peptide sequence homologous to the catalytic domain of proteins transferring sugars. Finally, the last exon encodes a domain homologous to the SH3 conserved motif of the SH2-SH3 protein family. This organization suggests that intramolecular interactions might give a tulip-shaped scaffolding, including the catalytic pocket of the enzyme in the Golgi lumen. Deduced from the published sequence of chromosome 14 (AL109847), the human gene organization of FUT8 seems to be similar to that of bovine FUT8b, although the exon partition is more pronounced (bovine exons 1 and 2 correspond to human exons 1-6). The mosaicism and phylogenetic positions of the alpha6-fucosyltransferase genes are compared with those of other fucosyltransferase genes.     

Achieving donor-specific hyporesponsiveness is associated with FOXP3+ regulatory T cell recruitment in human renal allograft infiltrates

Exploring new immunosuppressive strategies inducing donor-specific hyporesponsiveness is an important challenge in transplantation. For this purpose, a careful immune monitoring and graft histology assessment is mandatory. Here, we report the results of a pilot study conducted in twenty renal transplant recipients, analyzing the immunomodulatory effects of a protocol based on induction therapy with rabbit anti-thymocyte globulin low doses, sirolimus, and mofetil mycophenolate. Evolution of donor-specific cellular and humoral alloimmune response, peripheral blood lymphocyte subsets and apoptosis was evaluated. Six-month protocol biopsies were performed to assess histological lesions and presence of FOXP3+ regulatory T cells (Tregs) in interstitial infiltrates. After transplantation, there was an early and transient apoptotic effect, mainly within the CD8+ HLADR+ T cells, combined with a sustained enhancement of CD4+ CD25(+high) lymphocytes in peripheral blood. The incidence of acute rejection was 35%, all steroid sensitive. Importantly, only pretransplant donor-specific cellular alloreactivity could discriminate patients at risk to develop acute rejection. Two thirds of the patients became donor-specific hyporesponders at 6 and 24 mo, and the achievement of this immunologic state was not abrogated by prior acute rejection episodes. Remarkably, donor-specific hyporesponders had the better renal function and less chronic renal damage. Donor-specific hyporesponsiveness was inhibited by depleting CD4+ CD25(+high) T cells, which showed donor-Ag specificity. FOXP3+ CD4+ CD25(+high) Tregs both in peripheral blood and in renal infiltrates were higher in donor-specific hyporesponders than in nonhyporesponders, suggesting that the recruitment of Tregs in the allograft plays an important role for renal acceptance. In conclusion, reaching donor-specific hyporesponsiveness is feasible after renal transplantation and associated with Treg recruitment in the graft.     

DNA interaction and antiproliferative behavior of the water soluble platinum supramolecular squares [(en)Pt(N-N)]4(NO3)8 (en=ethylenediamine, N-N=4,4'-bipyridine or 1,4-bis(4-pyridyl)tetrafluorobenzene)

The interaction with DNA of two water soluble platinum supramolecular squares [(en)Pt(N-N)]4(NO3)8 (en=ethylenediamine, N-N=1,4-bis(4-pyridyl)tetrafluorobenzene, compound 1, N-N=4,4'-bipyridine, compound 2) has been studied by circular dichroism, electrophoretic mobility and atomic force microscopy. the two complexes drastically modify the second and tertiary structures of DNA, but compound 2 does it strongly due probably to its smaller size by comparison with compound 1 and its more suitable structural features for intercalation between base pairs. The two supramolecular squares were assayed against the HL-60 tumor cell line for 24 and 72 h. The IC50 values for 24 h are smaller than that of cisplatin for this time, however for 72 h the IC50 have higher values being the corresponding to compound 2 comparable to that of cisplatin. Apoptotic assays were also carried out for the compounds 1 and 2 against the tumor cell line.