Requirement of the Lec35 gene for all known classes of monosaccharide-P-dolichol-dependent glycosyltransferase reactions in mammals

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-beta-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.     

Development of Dot-ELISA for the detection of human rotavirus antigen and comparison with RNA-PAGE

AIMS: Development of a simple, specific, rapid and inexpensive Dot-ELISA test for diagnosis of rotaviral antigen in stool samples. METHODS AND RESULTS: Hyperimmune rabbit antisera raised against SA-11 (Simian Agent-11) strain was used as primary antibody. The secondary antibody conjugate used was the goat anti-rabbit IgG alkaline phosphatase, and BCIP/NBT solution was used as substrate. Faecal extracts were diluted 10-fold and used for the detection of rotavirus antigen. RNA-PAGE was performed to compare the specificity and sensitivity of the diagnostic tests. Dot-ELISA positive samples were further confirmed by Western blot analysis. CONCLUSIONS: This Dot-ELISA test could be used as an alternative method for diagnosing rotaviral samples in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: The Dot-ELISA test is simple, specific, rapid and cost effective. It is suitable for identifying a large number of samples obtained from epidemiological studies and hence, reducing the death rate of rotavirus-infected patients.     

Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB

The crystal structure of the unphosphorylated state of methylesterase CheB shows that the regulatory domain blocks access of substrate to the active site of the catalytic domain. Phosphorylation of CheB at Asp56 results in a catalytically active transiently phosphorylated enzyme with a lifetime of approximately two seconds. Solvent accessibility changes in this transiently phosphorylated state were probed by MALDI-TOF-detected amide hydrogen/deuterium exchange. No changes in solvent accessibility were seen in the regulatory domain upon phosphorylation of Asp56, but two regions in the catalytic domain (199-203 and 310-317) became more solvent accessible. These two regions flank the active site and contain domain-domain contact residues. Comparison with results from the isolated catalytic domain-containing C-terminal fragment of CheB (residues 147-349) showed that the increased solvent accessibility was less than would have occurred upon detachment of the regulatory domain. Thus, phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of CheB.     

Intracellular neuronal calcium sensor (NCS) protein VILIP-1 modulates cGMP signalling pathways in transfected neural cells and cerebellar granule neurones

The family of intracellular neuronal calcium-sensors (NCS) belongs to the superfamily of EF-hand proteins. Family members have been shown by in vitro assays to regulate signal cascades in retinal photoreceptor cells. To study the functions of NCS proteins not expressed in photoreceptor cells we examined Visinin-like protein-1 (VILIP-1) effects on signalling pathways in living neural cells. Visinin-like protein-1 expression increased cGMP levels in transfected C6 and PC12 cells. Interestingly, in transfected PC12 cells stimulation was dependent on the subcellular localization of VILIP-1. In cells transfected with membrane-associated wild-type VILIP-1 particulate guanylyl cyclase (GC) was stimulated more strongly than soluble GC. In contrast, deletion of the N-terminal myristoylation site resulted in cytosolic localization of VILIP-1 and enhanced stimulation of soluble GC. To study the molecular mechanisms underlying GC stimulation VILIP-1 was examined to see if it can physically interact with GCs. A direct physical interaction of VILIP-1 with the recombinant catalytic domain of particulate GCs-A, B and with native GCs enriched from rat brain was observed in GST pull-down as well as in surface plasmon resonance interaction studies. Furthermore, following trituration of recombinant VILIP-1 protein into cerebellar granule cells the protein influenced only signalling by GC-B. Together with the observed colocalization of GC-B, but not GC-A, with VILIP-1 in cerebellar granule cells, these results suggest that VILIP-1 may be a physiological regulator of GC-B.     

5'-Phosphodiesterase (5'-PDE) from germinated barley for hydrolysis of RNA to produce flavour nucleotides

5'-Phosphodiesterase (5'-PDE) is an enzyme that hydrolyses RNA to a mixture of ribonucleotides, from which the flavour enhancers, 5'-guanosine monophosphate (5'-GMP) and 5'-inosine monophosphate (5'-IMP) can be isolated. In the present work, 5'-PDE was extracted and partially purified from germinated barley seeds. 5'-PDE activity was monitored using bis-p-nitrophenyl phosphate as the substrate. The enzyme acts on the substrate and releases the p-nitrophenol, which is measured at 420 nm. Ultrafiltration using a polysulfone membrane having molecular weight cut off (MWCO) of 20 kDa gave 12-fold concentration. Further purification using ammonium sulphate gave 18-fold concentration. Heat shock for 15 min at 60 degrees C after the ultrafiltration enhanced the concentration of 5'-PDE 9.10 fold, while a similar treatment after ammonium sulphate treatment enhanced it by 17.83-fold. The enzyme had a pH optimum of 5, and was stable at 0 degrees C. This partially purified enzyme could be used for hydrolysis of RNA to produce 5'-GMP and 5' adenosine monophosphate, a precursor of 5'-IMP.     

Size-exclusion chromatography in multidimensional separation schemes for proteome analysis

Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), well-established techniques relying upon similar criteria for separation.Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed.     

Topoisomerase II antagonism and anticancer activity of coordinated derivatives of [RuCl(2)(C(6)H(6))(dmso)

Topoisomerase II poisoning and anticancer activity by the organometallic compound [RuCl(2)(C(6)H(6))(dmso)] was shown by us in an earlier study [Biochemistry 38 (1999) 4382]. Since high concentrations of this complex were required to achieve either effects, we have synthesized four derivatives of this complex in which the dimethyl sulphoxide group on the ruthenium atom was replaced with pyridine, 3-aminopyridine, p-aminobenzoic acid, and aminoguanidine. Three of these molecules showed enhanced potency of topoisomerase II poisoning and consequently also showed higher anticancer activity in breast and colon carcinoma cells in vitro. Detailed analysis of the molecular action of these compounds on topoisomerase II activity was carried out using the classical relaxation and cleavage activity of the enzyme, which revealed that the compounds poison topoisomerase II by freezing the enzyme and enzyme-cleaved DNA in a ternary "cleavage complex". The cleavage complex is implicated in the anti-neoplastic activity of these compounds. DNA interaction studies showed that these compounds interact with DNA in much the same way as [RuCl(2)(C(6)H(6))(dmso)], by external binding of the DNA helix. This is unlike most other topoisomerase II poisons, which predominantly interact with DNA through intercalation with the double helix.     

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, M(pro), a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus M(pro) is reported. The structure was refined to 1.96 A resolution and revealed three dimers in the asymmetric unit. The mutual arrangement of the protomers in each of the dimers suggests that M(pro) self-processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin-like fold that is connected by a 16 residue loop to an extra domain featuring a novel alpha-helical fold. Molecular modelling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of M(pro) substrates. Interactions involving the N-terminus and the alpha-helical domain stabilize the loop in the orientation required for trans-cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.     

Transcriptome analysis of the retina

The retina offers unique opportunities to define the molecular and cellular pathways mediating neuronal function and disease because of its morphological complexity, well-defined role in visual transduction and the availability of mutants. These investigations are being greatly facilitated by the ongoing identification of genes expressed in the retina using high-throughput methods.