Mineo Saneyoshi

Isolation and Characterization of Rhamnose-binding Lectins from Eggs of Steelhead Trout (Oncorhynchus mykiss) Homologous to Low Density Lipoprotein Receptor Superfamily

Two l-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatography and ion exchange chromatography. The apparent molecular masses of purified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively, by gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was noncovalently linked trimer of 21.5-kDa subunits. The minimum concentrations of STL1, a major component, and STL2, a minor component, needed to agglutinate rabbit erythrocytes were 9 and 0.2 μg/ml, respectively. The most effective saccharide in the hemagglutination inhibition assay for both STL1 and STL2 was l-rhamnose. Saccharides possessing the same configuration of hydroxyl groups at C2 and C4 as that in l-rhamnose, such asl-arabinose and d-galactose, also inhibited. The amino acid sequence of STL2 was determined by analysis of peptides generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I orStaphylococcus aureus V8 protease. The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture; that is, it was composed of two tandemly repeated homologous domains (STL2-N and STL2-C) with 52% internal homology. These two domains showed a sequence homology to the subunit (105 amino acid residues) ofd-galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively). The N terminus of the STL1 subunit was blocked with an acetyl group. However, a partial amino acid sequence of the subunit showed a sequence similarity to STL2. Moreover, STL2 also showed a sequence homology to the ligand binding domain of the vitellogenin receptor. We have also employed surface plasmon resonance biosensor methodology to investigate the interactions between STL2 and major egg yolk proteins from steelhead trout, lipovitellin, and β′-component, which are known as vitellogenin digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (K a ) of STL2 to lipovitellin and β′ component were 3.44 × 106 and 4.99 × 106, respectively. These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor super- family.

Presumed anticodon structure of glutamic acid tRNA from E. coli: A possible location of a 2-thiouridine derivative in teh first position of the anticodon

Abstract Carbon et al. previously reported isolation of 5-methylaminomethyl-2-thiouridine from unfractionated E. coli tRNA as a new minor component (1). However, its exact location in the nucleotide sequence of a particular tRNA is not yet known. In addition, its biological role in tRNA is not understood. This paper reports that a 2-thiouridine derivative, tentatively characterized as 5-methylaminomethyl-2-thiouridine (referred to as N hereafter) was isolated from purified E. coli tRNA2Glu and sequence analysis of the oligonucleotide containing N revealed that this oligonucleotide was possibly the anticodon loop and this minor nucleoside was located in the first position of the anticodon.

Rhamnose-binding lectins from steelhead trout (Oncorhynchus mykiss) eggs recognize bacterial lipopolysaccharides and lipoteichoic acid.

The interaction between bacteria and three L-rhamnose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces.

The presence of N-[9-(β-D-ribofuranosyl)purin-6-ylcarbamoyl]threonine in serine, methionine and lysine transfer RNA's from Escherichia coli

The isolation and characterization of N-[9-(β-D-ribofuranosyl)-purin-6-ylcarbamoyl]threonine from tRNA3Ser, tRNA1Met and tRNALys of E. coli is described. The specific role of this minor component of tRNA in the recognition of codons starting with A is discussed.

Immunohistochemical localization of rhamnose-binding lectins in the steelhead trout (Oncorhynchus mykiss)

The localization of three -rhamnose-binding lectins named STL1, STL2, and STL3 from eggs of steelhead trout (Oncorhynchus mykiss) was analyzed by indirect immunohistochemical staining with specific antisera against individual lectins. In early previtellogenic oocyte, STL1 was localized not only in the cortical vesicles, but also in the plasma membrane and germinal vesicle. On the other hand, STL2 and STL3 were localized only in the cortical vesicles. In pre-fertilization mature egg, STLs were localized in a thin layer of cortical granules at the cytoplasmic side of the plasma membrane. STLs were accumulated on the surface of cytoplasm and inner membrane 30 min after fertilization. The strong staining with anti-STL1 antiserum was observed in several tissues and cells of the steelhead trout, such as spleen, thrombocytes, and blood leukocytes, but not erythrocytes. STL1 was also identified in exocrine cells, such as goblet cells of intestine and mucous cells of gill. These results indicate that the multiple lectins found in eggs of the steelhead trout play physiological roles not only in eggs, but also in various cells related to the innate immunity.

Isolation and characterization of N6-methyladenosine from Escherichia coli valine transfer RNA

Abstract A new minor component has been isolated from Escherichia coli tRNAValI which was identified as 6-methylamino-9-β- d -ribofuranosylpurine (N6-methyladenosine). Precautions were taken against possible conversion of N1-methyladenosine or N6-isopentenyladenosine to N6-methyladenosine during the isolation of the tRNAValI and subsequent liberation of the minor component from the tRNA. The N6-methyladenylic acid obtained by mild ribonuclease treatment of the tRNA was subsequently converted to the corresponding nucleoside and to the base by treatment with E. coli alkaline phosphatase and acid hydrolysis, respectively. The identity of the nucleotide, nucleoside and base thus obtained with corresponding authentic samples was established by comparison of their ultraviolet absorption spectra, paper chromatographic mobilities in several solvent systems, and by low- and high-resolution mass spectra.

A Novel Rhamnose-binding Lectin Family from Eggs of Steelhead Trout (Oncorhynchus mykiss) with Different Structures and Tissue Distribution

An L-rhamnose-binding isolectin named STL3 (subunit Mr, 21.5 k) was isolated from eggs of the steelhead trout (Oncorhynchus mykiss) in addition to STL1 (subunit Mr, 31.4 k) and STL2 (subunit Mr, 21.3 k) that had been already isolated. STLs were composed of non-covalently linked subunits. The primary structures of STL1 and STL3 were analyzed by the combined use of protein sequencing and cDNA sequencing. A cDNA encoding STL2, of which the protein sequence had been previously studied, was also analyzed. The STL1 subunit (289 amino acid residues) had different structural properties compared to those of the STL2 subunit (195 amino acid residues) and the STL3 subunit (195 amino acid residues); e.g., the number of repeated domain (three for STL1, and two for STL2 and STL3), although all of them were composed of tandemly repeated homologous domains (40 to 53% identities). The lectin levels in various tissues and during the embryonic development showed that STL1 had different distribution and expression profiles from those of STL2 and STL3. Although STL1 could be detected in several tissues and serum of both male and female steelhead trout, STL2 and STL3 were only abundant in the ovary. STL2 and STL3 levels dramatically decreased just after hatching, however, the STL1 level increased temporarily. These results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.

Dehydroaltenusin, a Mammalian DNA Polymerase α Inhibitor

Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase α (pol α) in vitro. Surprisingly, among the polymerases and DNA metabolic enzymes tested, dehydroaltenusin inhibited only mammalian pol α. Dehydroaltenusin did not influence the activities of the other replicative DNA polymerases, such as δ and ε; it also showed no effect even on the pol α activity from another vertebrate (fish) or plant species. The inhibitory effect of dehydroaltenusin on mammalian pol α was dose-dependent, and 50% inhibition was observed at a concentration of 0.5 μm. Dehydroaltenusin-induced inhibition of mammalian pol α activity was competitive with the template-primer and non-competitive with the dNTP substrate. BIAcore analysis demonstrated that dehydroaltenusin bound to the core domain of the largest subunit, p180, of mouse pol α, which has catalytic activity, but did not bind to the smallest subunit or the DNA primase p46 of mouse pol α. These results suggest that the dehydroaltenusin molecule competes with the template-primer molecule on its binding site of the catalytic domain of mammalian pol α, binds to the site, and simultaneously disturbs dNTP substrate incorporation into the template-primer.

Selective inactivation of amino acid acceptor and ribosome-binding activities of Escherichia coli tRNA by modification with cyanogen bromide

Abstract 1. Modification of Escherichia coli tRNA with cyanogen bromide caused a selective loss of acceptor activities for glutamic acid, lysine and glutamine. However, most tRNAs known to contain 4-thiouridine, which is susceptible to cyanogen bromide, retained their amino acid acceptor activities after the modification, indicating that modification of the 4-thiouridylate residue has no effect on amino acid acceptor activity. 2. Inactivation of glutamic acid acceptor activity was due to a modification of 5-methylaminomethyl-2-thiouridine located in the first position of the anticodon of tRNA Glu , showing that the anticodon region is related to the recognition site of aminoacyl-tRNA synthetase in this particular tRNA. 3. The binding abilities of [ 14 C]tyrosyl-, [ 14 C]histidyl- and [ 14 C]lysyl-tRNAs to ribosomes were specifically inactivated by the modification, whereas those of [ 14 C]arginyl-, [ 14 C]isoleucyl-, [ 14 C]glycyl-, [ 14 C]phenylalanyl- and [ 14 C]valyl-tRNA were not affected. It is likely that the inactivation of the ribosome-binding ability of tRNA is due to the modification of other minor nucleosides which are susceptible to cyanogen bromide rather than to modification of 4-thiouridine. 4. [ 14 C]Tyrosyl-tRNA modified with CNBr formed a complex with poly (U 4 ,G) in the absence of ribosomes. This suggested that modification with cyanogen bromide caused a distortion of the conformational structure of tRNA Tyr .

Uridin-5-oxy acetic acid: A new minor constituent from E, coli valine transfer RNA I

Abstract The primary sequence of E , coli tRNAIVal was recently established (1,2), and it was found that an unidentified minor component designated as V was located in the first position of the anticodon of this tRNA (1–3). The unique structure of V is of particular interest, since it must participate directly in codon-anticodon base pairing in the decoding process in protein synthesis. This report describes the characterization of this minor nucleoside as uridin-5-oxy acetic acid (Fig. 1).