The sequence of the mouse 14 kDa beta-galactoside-binding lectin and evidence for its synthesis on free cytoplasmic ribosomes.
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ABSTRACT:
SUBMITTER: Wilson TJ
PROVIDER: S-EPMC1138908 | biostudies-other | 1989 Aug
REPOSITORIES: biostudies-other
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Project description:A full-length cDNA clone for the 14 kDa soluble beta-galactoside-binding lectin of man has been isolated from a cDNA library from HepG2 hepatoma cells. The derived amino acid sequence is identical with that of the 14 kDa lectin from human placenta. The results of Northern and Southern blotting of several different human cell lines using a cDNA probe for the 14 kDa lectin suggest the presence of a single gene for this protein. Thus, although there are multiple proteins in the range 14-200 kDa which are antigenically related to this lectin, we would conclude from the present study that there is only one gene for the 14 kDa lectin.
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Project description:The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.
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Project description:Pseudouridine (?) is present at conserved, functionally important regions in the ribosomal RNAs (rRNAs) from all three domains of life. Little, however, is known about the functions of ? modifications in bacterial ribosomes. An Escherichia coli strain has been constructed in which all seven rRNA ? synthases have been inactivated and whose ribosomes are devoid of all ?s. Surprisingly, this strain displays only minor defects in ribosome biogenesis and function, and cell growth is only modestly affected. This is in contrast to a strong requirement for ? in eukaryotic ribosomes and suggests divergent roles for rRNA ? modifications in these two domains.IMPORTANCE Pseudouridine (?) is the most abundant posttranscriptional modification in RNAs. In the ribosome, ? modifications are typically located at conserved, critical regions, suggesting they play an important functional role. In eukarya and archaea, rRNAs are modified by a single pseudouridine synthase (PUS) enzyme, targeted to rRNA via a snoRNA-dependent mechanism, while bacteria use multiple stand-alone PUS enzymes. Disruption of ? modification of rRNA in eukarya seriously impairs ribosome function and cell growth. We have constructed an E. coli multiple deletion strain lacking all ? modifications in rRNA. In contrast to the equivalent eukaryotic mutants, the E. coli strain is only modestly affected in growth, decoding, and ribosome biogenesis, indicating a differential requirement for ? modifications in these two domains.
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