RESUMO
Toxic lectins of European mistletoe Viscum album L.--MLI (viscumin), MLII and MLIII--are present in water extracts of this plant. Earlier we have cloned the full-length gene of MLIII precursor [A.G. Tonevitsky, I.I. Agapov, I.B. Pevzner, N.V. Maluchenko, M.M. Mojsenovich, U. Pfueller, M.P. Kirpichnikov, (2004) Biochemistry (Mosc.), 69 (6), 790-800, in press]. Here for the first time we report the cloning and expression in Escherichia coli cells of MLIII gene fragment encoding the carbohydrate-binding subunit. We have proved with our panel of monoclonal antibodies against ML toxins that the cloned fragment encoded MLIII B-subunit. The immunochemical and sugar-binding activities of renatured recombinant MLIII B-subunit were demonstrated in ELISA and ELLA, respectively. The comparative analysis of amino acid sequences of the cloned rMLIIIB and the B-subunits of other type II RIPs--MLI, ricin, abrin and nigrin b--was performed, revealing the main differences in primary structure of MLI and MLIII B-chains, which could determine their sugar specificity. The antigenicity analysis of MLI and MLIII B-subunits showed one epitope 25RDDDFRDGNQ34 in MLIB that is absent in MLIIIB sequence. The role of the toxic lectins and their subunits in immunological properties of mistletoe extracts is discussed.
Assuntos
Metabolismo dos Carboidratos , Epitopos , Erva-de-Passarinho/química , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Adjuvantes Imunológicos , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Erva-de-Passarinho/genética , Dados de Sequência Molecular , Preparações de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2 , Ricina/genética , Ricina/metabolismo , Homologia de Sequência de Aminoácidos , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Viscum album/químicaRESUMO
The X-ray structure of mistletoe lectin I (MLI), a type-II ribosome-inactivating protein (RIP), cocrystallized with galactose is described. The model was refined at 3.0 A resolution to an R-factor of 19.9% using 21 899 reflections, with Rfree 24.0%. MLI forms a homodimer (A-B)2 in the crystal, as it does in solution at high concentration. The dimer is formed through contacts between the N-terminal domains of two B-chains involving weak polar and non-polar interactions. Consequently, the overall arrangement of sugar-binding sites in MLI differs from those in monomeric type-II RIPs: two N-terminal sugar-binding sites are 15 A apart on one side of the dimer, and two C-terminal sugar-binding sites are 87 A apart on the other side. Galactose binding is achieved by common hydrogen bonds for the two binding sites via hydroxy groups 3-OH and 4-OH and hydrophobic contact by an aromatic ring. In addition, at the N-terminal site 2-OH forms hydrogen bonds with Asp27 and Lys41, and at the C-terminal site 3-OH and 6-OH undergo water-mediated interactions and C5 has a hydrophobic contact. MLI is a galactose-specific lectin and shows little affinity for N-acetylgalactosamine. The reason for this is discussed. Structural differences among the RIPs investigated in this study (their quaternary structures, location of sugar-binding sites, and fine sugar specificities of their B-chains, which could have diverged through evolution from a two-domain protein) may affect the binding sites, and consequently the cellular transport processes and biological responses of these toxins.
Assuntos
Adjuvantes Imunológicos/química , Galactose/química , Erva-de-Passarinho/química , Preparações de Plantas/química , Proteínas de Plantas , Toxinas Biológicas/química , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Galactose/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Preparações de Plantas/metabolismo , Conformação Proteica , Proteínas Inativadoras de Ribossomos Tipo 2 , Alinhamento de Sequência , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismoRESUMO
Ricin and viscumin are heterodimeric protein toxins. Their A-chain is enzymatically active and removes an adenine residue from the 28S rRNA, the B-chain has lectin activity and binds to terminal galactose residues of cell surface receptors. The toxins reveal a high degree of identity in their amino acid sequences. Nevertheless, uptake into 3T3 cells occurs via different receptors and endocytotic pathways. This has been revealed by enzyme linked based analysis of ricin competition with viscumin, and by fluorochrome-labeled toxins (viscumin-FITC, ricin-Alexa 568), which were added simultaneously or separately to living cells. Then the uptake was followed by confocal laser scanning microscopy. Ricin immediately is delivered to the tubular and vesicular structures of endosomes in the perinuclear area while viscumin becomes endocytosed into small vesicles preferentially in the cell periphery. After about 60 min both these toxins may be found in tubo-vesicular structures of endosomes where the sorting process can directly be observed. The fact that this sorting takes place is a strong argument for the assumption that the toxins are bound to membrane proteins, either to their original receptors or to other proteins inside the endosomal compartment exhibiting terminal galactose residues. The toxins are biologically fully active as has been proven by binding and by toxicity experiments, thus the differences in targeting do not arise from labeling.