Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H

The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half-lives of SIB1 and E. coli RNases HII at 30 degrees C were approximately 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by approximately 0.2 M and approximately 5 degrees C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 degrees C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low- and high-activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high- and low-activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with RNase H families.     

Cloning and expression in Escherichia coli of a xylanase-encoding gene from the yeast Cryptococcus albidus.

In the yeast, Cryptococcus albidus, a comparison between the sequence of the xylanase (XLN)-encoding chromosomal gene (XLN) and the cDNA sequence reveals the presence of seven introns, ranging in length from 51 to 69 bp. One of their 5' splice site sequences is similar to the consensus sequence for yeast, while the other six resemble the consensus sequence for higher eukaryotes. Their 3' end splice site sequences are representative of the conserved sequence found in eukaryotes. Their putative branching point sequences are different from the well-known conserved sequence, 5'-TACTAAC, observed in yeast, but again resemble the mammalian one. The cDNA encoding XLN is expressed by Escherichia coli, under the control of the lacZ promoter. The gene product remains inside the cell and has a molecular size of 40 kDa, which matches the size of the nonglycosylated protein. When compared to the glycosylated enzyme, the nonglycosylated XLN from E. coli shows twofold less affinity for substrate and its Vmax is 100-fold lower. Moreover, the nonglycosylated XLN only acts on large xylan polymers and very slightly on xylohexaose.     

RNase T affects Escherichia coli growth and recovery from metabolic stress.

To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed.     

Rapid identification of viable Escherichia coli subspecies with an electrochemical screen-printed biosensor array

Rapid identification of Escherichia coli strains is an important diagnostic goal in applied medicine as well as the environmental and food sciences. This paper reports an electrochemical, screen-printed biosensor array, where selective recognition is accomplished using lectins that recognize and bind to cell-surface lipopolysaccharides and coulometric transduction exploits non-native external oxidants to monitor respiratory cycle activity in lectin-bound cells. Ten different lectins were separately immobilized onto porous membranes that feature activated surfaces (ImmunodyneABC). Modified membranes were exposed to untreated E. coli cultures for 30 min, rinsed, and layered over the individual screen-printed carbon electrodes of the sensor array. The membranes were were incubated 5 min in a reagent solution that contained the oxidants menadione and ferricyanide as well as the respiratory substrates succinate and formate. Electrochemical oxidation of ferrocyanide for 2 min provided chronocoulometric data related to the quantities of bound cells. These screen-printed sensor arrays were used in conjunction with factor analysis for the rapid identification of four E. coli subspecies (E. coli B, E. coli Neotype, E. coli JM105 and E. coli HB101). Systematic examination of lectin-binding patterns showed that these four E. coli subspecies are readily distinguished using only five essential lectins.     

Human non-secretory ribonucleases. I. Purification, peptide mapping and lectin blotting analysis of the kidney, liver and spleen enzymes

Human non-secretory neutral ribonucleases (RNases) from kidney,liver and spleen have been purified and characterized. SDS—PAGEindicates that all three RNases are highly purified and haveapparent mol. wts of 17–18 kDa. Kinetic analysis indicatesthat all three RNases have a broad pH optimum centred around6.5, and all three have similar substrate specificities withsignificant preference for RNA and poly(U) when compared topoly(C), poly (A) and poly(G). All of the above data, as wellas immunoblotting data using three polyclonal antibodies (anti-humanliver RNase, anti-human pancreatic RNase, anti-human eosino-phil-derivedneurotoxin), indicate that the three proteins are highly purifiedand are non-secretory RNases (IIN). Further characterizationby cyanogen bromide peptide mapping and extensive lectin blottingindicated no significant differences between the three humanRNases. All three RNases appear to have very similar, if notidentical, protein backbones and all three are glycoproteinswhich are recognized by lectins with specificity for GlcNAc,Fuc and, to a lesser extent, with specificity for Galß(1–4)GlcNAc.No significant tissuespecific differences were found among thethree human non-secretory RNases. lectin blotting non-secretory RNases peptide mapping     

Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB).

The human cytomegalovirus (HCMV) envelope glycoprotein complex gp55-116 was expressed in both Escherichia coli and cells infected with a recombinant vaccinia virus. E. coli produced a single protein of Mr 100,000 which approximated the size of the nonglycosylated gp55-116 precursor found in HCMV-infected cells. Cells infected with the recombinant vaccinia virus contained three intracellular forms of Mr 160,000, 150,000, and 55,000 which were detected by a monoclonal antibody reactive with gp55. Comparison of the immunological properties of these recombinant proteins indicated that several of the HCMV gp55-116 monoclonal antibodies and sera from patients infected with HCMV reacted with the vaccinia virus-derived proteins whereas a more restricted group of monoclonal antibodies recognized the E. coli-produced protein. Immunization of mice with either E. coli or vaccinia virus recombinant HCMV gp55-116 resulted in production of virus-neutralizing antibodies. In contrast to the almost exclusive production of complement-dependent neutralizing antibodies following immunization with recombinant vaccinia virus, the E. coli-derived protein induced complement-independent neutralizing antibodies.     

Tetranectin, a trimeric plasminogen-binding C-type lectin.

Tetranectin, a plasminogen-binding protein belonging to the family of C-type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin-as well as natural tetranectin from human plasma-was shown by chemical cross-linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization.     

A Shared Endoplasmic Reticulum-associated Degradation Pathway Involving the EDEM1 Protein for Glycosylated and Nonglycosylated Proteins

Studies of misfolded protein targeting to endoplasmic reticulum-associated degradation (ERAD) have largely focused on glycoproteins, which include the bulk of the secretory proteins. Mechanisms of targeting of nonglycosylated proteins are less clear. Here, we studied three nonglycosylated proteins and analyzed their use of known glycoprotein quality control and ERAD components. Similar to an established glycosylated ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a, its nonglycosylated mutant, makes use of calnexin, EDEM1, and HRD1, but only glycosylated H2a is a substrate for the cytosolic SCF^Fbs2 E3 ubiquitin ligase with lectin activity. Two nonglycosylated BiP substrates, NS-1κ light chain and truncated Igγ heavy chain, interact with the ERAD complex lectins OS-9 and XTP3-B and require EDEM1 for degradation. EDEM1 associates through a region outside of its mannosidase-like domain with the nonglycosylated proteins. Similar to glycosylated substrates, proteasomal inhibition induced accumulation of the nonglycosylated proteins and ERAD machinery in the endoplasmic reticulum-derived quality control compartment. Our results suggest a shared ERAD pathway for glycosylated and nonglycosylated proteins composed of luminal lectin machinery components also capable of protein-protein interactions.     

Effects of growth phase and boiling of enteropathogenic Escherichia coli strains on their interaction with Pseudomonas aeruginosa lectins

Escherichia coli strains from' serotypes O86, 0128 and O111 varied in their reactivity with Pseudomonas aeruginose lectins (PA-I with D-galactose specificity and PA-II which binds L-fucose, D-mannose, L-galactose and D-fructose). Generally, cells of O86 strains were agglutinated by PA-I, but not by PA-II, and those of O128 serotype were agglutinated by PA-II, and not by PA-I. Adsorption tests showed that cells of E. coli O86 strains adsorb PA-I to a greater extent than PA-II, while most E. coli O128 strains adsorbed higher amounts of PA-II. Cells of E. coli O111B4 which were not agglutinated by either Pseudomonas lectin could still adsorb both. Boiling of O86 and O128 cells frequently enhanced their agglutinability as well as their lectin adsorption capacity. The agglutinability enhancement was somewhat more prominent in boiled stationary phase cells than in log phase cells probably due to late synthesis of the O antigen components concomitantly with the heat-sensitive components (K antigens) which masked them. PA-I agglutinating activity was inhibited by the lipopolysaccharide (LPS) extracted from E. coli O86 cells, while PA-II was inhibited by the LPS extracted from E. coli O128 cells. These findings indicate that the receptors to the Pseudomonas lectins probably reside in the terminal part of the O-specific-polysaccharide of the LPSs of these bacteria.     

Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

Summary We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium.     

Structural, thermodynamic, and mutational analyses of a psychrotrophic RNase HI

Ribonuclease (RNase) HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 was overproduced in Escherichia coli, purified, and structurally and biochemically characterized. The amino acid sequence of MR-1 RNase HI is 67% identical to that of E. coli RNase HI. The crystal structure of MR-1 RNase HI determined at 2.0 A resolution was highly similar to that of E. coli RNase HI, except that the number of intramolecular ion pairs and the fraction of polar surface area of MR-1 RNase HI were reduced compared to those of E. coli RNase HI. The enzymatic properties of MR-1 RNase HI were similar to those of E. coli RNase HI. However, MR-1 RNase HI was much less stable than E. coli RNase HI. The stability of MR-1 RNase HI against heat inactivation was lower than that of E. coli RNase HI by 19 degrees C. The conformational stability of MR-1 RNase HI was thermodynamically analyzed by monitoring the CD values at 220 nm. MR-1 RNase HI was less stable than E. coli RNase HI by 22.4 degrees C in Tm and 12.5 kJ/mol in DeltaG(H2O). The thermodynamic stability curve of MR-1 RNase HI was characterized by a downward shift and increased curvature, which results in an increased DeltaCp value, compared to that of E. coli RNase HI. Site-directed mutagenesis studies suggest that the difference in the number of intramolecular ion pairs partly accounts for the difference in stability between MR-1 and E. coli RNases HI.     

Escherichia coli rna gene encoding RNase I: cloning, overexpression, subcellular distribution of the enzyme, and use of an rna deletion to identify additional RNases.

The cloning and overexpression of the Escherichia coli rna gene encoding RNase I are described. Only a single copy of the rna gene is present on the E. coli chromosome. Although cells with as much as a 100-fold increase in RNase I activity were constructed, little effect on cell growth was observed. Overexpressed RNase I was found in the periplasmic space to the same degree (approximately 85%) as wild-type enzyme, suggesting no limitation in RNase I transport. The rna clone was used to identify a deletion strain totally lacking the rna gene. The normal growth of this strain showed that RNase I is not essential for cell viability. Extracts from the RNase I deletion strain still retained a low level of RNase activity in the presence of EDTA, conclusively demonstrating the existence of additional EDTA-active RNases in E. coli. The possibility of a RNase I inhibitor is also discussed.     

P-type lectins

The two members of the P-type lectin family, the cation-dependent mannose 6-phosphate receptor (CD-MPR) and the insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/MPR), are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The P-type lectins play an essential role in the generation of functional lysosomes within the cells of higher eukaryotes by directing newly synthesized lysosomal enzymes bearing the mannose 6-phosphate (M6P) signal to lysosomes. At the cell surface, the IGF-II/MPR also binds to the nonglycosylated polypeptide hormone, IGF-II, targeting this potent mitogenic factor for degradation in lysosomes. Moreover, in recent years, the multifunctional nature of the IGF-II/MPR has become increasingly apparent, as the list of extracellular ligands recognized by this receptor has grown to include a diverse spectrum of M6P-containing proteins as well as nonglycosylated ligands, implicating a role for the IGF-II/MPR in a number of important physiological pathways. Recent investigations have provided valuable insights into the molecular basis of ligand recognition by the MPRs as well as the complex intracellular trafficking pathways traversed by these receptors. This review provides a current view on the structures, functions, and medical relevance of the P-type lectins.     

On the track of antitumour ribonucleases

Ribonucleases (RNases) are potential alternatives to non-mutagenic antitumour drugs. Among these enzymes, onconase, bovine-seminal ribonuclease and the Rana catesbeiana and Rana japonica lectins exert a cytotoxic activity that is selective for tumour cells. A model for the mechanism of cytotoxicity of these RNases which involves different steps is generally accepted. The model predicts that cytotoxicity requires interaction of the RNases with the cell membrane and internalisation to occur by endocytosis. Then, at a precise point, the RNases are translocated to the cytosol where they cleave cellular RNA if they have been able to preserve their ribonucleolytic activity. The cleavage of cellular RNA induces apoptosis but there is evidence suggesting that RNase-triggered apoptosis does not entirely result from the inhibition of protein synthesis. How efficiently a particular RNase carries out each of the steps determines its potency as a cytotoxin.     

The site of interaction of aminoacyl-tRNA with elongation factor Tu

We have used RNases T1, T2 and A to digest two aminoacyl-tRNAs, Escherichia coli Phe-tRNAPhe and E. coli Met- tRNAMetm both in the naked forms and in ternary complexes with E. coli elongation factor Tu (EF-Tu) and GTP. An analysis of the 'footprinting' results has led to an interpretation that has localized the part of the three-dimensional structure of aminoacyl-tRNA covered by the protein in the ternary complex. In terms of the three-dimensional structure of tRNA established for yeast tRNAPhe, EF-Tu covers the aa-end, aa-stem, T-stem, and extra loop on the side of the L-shaped tRNA that exposes the extra loop.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis.

The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA-RNA hybrid as a substrate revealed that the enzymatic properties of SIB1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was approximately 25 degrees C for SIB1 RNase HI and approximately 60 degrees C for E.coli RNase HI. The optimum temperature for the SIB1 RNase HI activity was also shifted downward by 20 degrees C compared with that of E.coli RNase HI. Nevertheless, SIB1 RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10 degrees C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the alphaI-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.     

A family 2a carbohydrate-binding module suitable as an affinity tag for proteins produced in Pichia pastoris

The family 2a carbohydrate-binding module (CBM), Cel5ACBM2a, from the C-terminus of Cel5A from Cellulomonas fimi, and Xyn10ACBM2a, the family 2a CBM from the C-terminus of Xyn10A from C. fimi, were compared as fusion partners for proteins produced in the methylotrophic yeast Pichia pastoris. Gene fusions of murine stem-cell factor (SCF) with both CBMs were expressed in P. pastoris. The secreted SCF-Xyn10ACBM2a polypeptides were highly glycosylated and bound poorly to cellulose. In contrast, fusion of SCF to Cel5ACBM2a, which lacks potential N-linked glycosylation sites, resulted in the production of polypeptides which bound tightly to cellulose. Cloning and expression of these CBM2a in P. pastoris without a fusion partner confirmed that N-linked glycosylation at several sites was responsible for the poor cellulose binding. The nonglycosylated CBMs produced in E. coli had very similar cellulose-binding properties.