Kiyoshi Tazaki

Sialylated Oligosaccharide-specific Plant Lectin from Japanese Elderberry (Sambucus sieboldiana) Bark Tissue Has a Homologous Structure to Type II Ribosome-inactivating Proteins, Ricin and Abrin cDNA CLONING AND MOLECULAR MODELING STUDY

Bark lectins from the elderberry species belonging to the genus Sambucus have a unique carbohydrate binding specificity for sialylated glycoconjugates containing NeuAc(α2-6)Gal/GalNAc sequence. To elucidate the structure of the elderberry lectin, a cDNA library was constructed from the mRNA isolated from the bark tissue of Japanese elderberry (Sambucus sieboldiana) with gt11 phage and screened with anti-S. sieboldiana agglutinin (SSA) antibody. The nucleotide sequence of a cDNA clone encoding full-length SSA (LecSSA1) showed the presence of an open reading frame with 1902 base pairs, which corresponded to 570 amino acid residues. This open reading frame encoded a signal peptide and a linker region (19 amino acid residues) between the two subunits of SSA, the hydrophobic (A-chain) and hydrophilic (B-chain) subunits. This indicates that SSA is synthesized as a preproprotein and post-translationally cleaved into two mature subunits. Homology searching as well as molecular modeling studies unexpectedly revealed that each subunit of SSA has a highly homologous structure to the galactose-specific lectin subunit and ribosome-inactivating subunit of plant toxic proteins such as ricin and abrin, indicating a close evolutionary relationship between these carbohydrate-binding proteins.

Occurrence of shikimic and quinic acids in angiosperms

Abstract Leaves of 83 angiosperms have been made surveyed for quinic and shikimic acids. The quinic acid content was higher in woody dicotyledons than in herbaceous dicotyledons or in the monocotyledons, substantiating the view that its presence may be correlated with the lignification in plants.

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark ofRobinia pseudoacacia

A cDNA clone encoding a lectin was isolated by immunological screening of an expression library prepared from poly(A)+ RNA from the inner bark ofRobinia pseudoacacia. The cDNA clone (RBL104) had an open reading frame of 858 bp that encoded a polypeptide with a predicted molecular weight of 31210. This molecular weight corresponded closely to that of a polypeptide immunoprecipitated from products of translationin vitro of the poly(A)+ RNA. Thus, RBL104 appeared to be a full-length cDNA. The N-terminal amino acid sequence of the purified lectin protein matched a portion of the predicted amino acid sequence. It appeared that the lectin was synthesized as a precursor that consisted of a putative signal peptide of 31 amino acids and a mature polypeptide of 255 amino acids. Southern blot analysis of the genomic DNA revealed that the lectin was encoded by a small multigene family. The lectin was mostly localized in the axial and ray parenchymal cells of the inner bark. A small amount of lectin was also found in the axial and ray parenchymal cells of the xylem. The lectin accumulated in the inner bark in September, remained at high levels during the winter and disappeared in May. The mRNA for the lectin was detected from August to the following March. The appearance and disappearance of the mRNA were observed prior to those of the lectin protein.

Studies by site-directed mutagenesis of the carbohydrate-binding properties of a bark lectin from Robinia pseudoacacia

A bark lectin, RBL, from Robinia pseudoacacia (black locust), binds galactose‐related sugars specifically. Recombinant RBL (rRBL) with a histidine tag was expressed in Escherichia coli, purified and characterized. rRBL agglutinated rabbit erythrocytes and the hemagglutination was inhibited by galactose and related sugars. To elucidate the mechanism of the binding of carbohydrate by RBL, 16 mutant rRBLs were produced by site‐directed mutagenesis. The analysis of the mutants indicated that residues Phe130 and Asp87 play key roles in the binding of carbohydrate by RBL. When Thr215, Leu217 and Ser218 in the carboxy‐terminal region were replaced by alanine, the respective replacements decreased the hemagglutinating activity. However, replacement by alanine of Glu219 did not decrease this activity. Three mutant rRBLs were generated by reference to the primary sequences of the proposed carbohydrate‐ and metal‐binding regions of mannose‐specific lectins. Although these rRBLs agglutinated rabbit erythrocytes, the hemagglutination was not inhibited by mannose. Substitution or insertion that yielded a partial sequence similar to those of l‐fucose‐specific lectins and hemagglutinin from Maackia amurensis resulted in a complete loss of the hemagglutinating activity of rRBL.© 1997 Federation of European Biochemical Societies.

Expression of cDNA for a bark lectin of Robinia in transgenic tobacco plants

A cDNA encoding a bark lectin of Robinia pseudoacacia was introduced into tobacco plants. The expression of the lectin cDNA under control of the 35S promoter was confirmed by Western blot analysis and a hemagglutination assay of extracts of transgenic plants. Western blot analysis revealed that the subunit of the lectin from tobacco had a molecular mass of 29 kDa. The sequence of nine amino acids from the N‐terminus of the lectin from transgenic tobacco plants was identical to that of the bark lectin from Robinia, indicating that the lectin had been processed correctly at its N‐terminus in tobacco. The molecular mass of the purified native lectin produced by tobacco plants was estimated to be 112 kDa by gel filtration on a column of Superdex 200. It is suggested that the lectin subunits assembled to form tetramers in transgenic tobacco plants.

Robinia pseudoacacia inner-bark lectin promoter expresses GUS also predominantly in phloem of transgenic tobacco

Summary Robinia pseudoacacia L., a leguminous deciduous tree, accumulates lectins in the inner bark (phloem) and seeds. The lectin gene Rplec2 is expressed predominantly in the inner bark. To examine the expression pattern directed by the 5′-upstream region (from -793 bp to +26 bp relative to the transcription start site) of Rplec2 in heterologous plants, a transcriptional fusion of this region with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants. The resulting transformants showed higher GUS activity in the stems than in the leaves and roots. Histochemical assay of GUS activity demonstrated that the expression of the GUS gene was confined to internal and external phloem, parenchyma cells adjacent to the phloem, and xylem parenchyma cells in the stems. In petioles, the GUS gene was expressed in phloem and nearby parenchyma cells, but not in leaf mesophyll cells. GUS activity was also detected in the phloem of the root. In addition, sequence analysis of the 5′-upstream region of Rplec2 revealed three conserved motifs shared with several previously reported phloem-specific promoters. These results indicate that the 5′-upstream region of Rplec2 functions as a promoter that directs preferential expression of the GUS gene to the phloem of transgenic tobacco plants.

Alicyclic acid metabolism in plants

When the epicotyls of etiolated pea seedlings were fed with 40 mM potassium quinate solution in the dark for 24 hr, a marked accumulation of shikimic acid occurred in the tissue. This effect was much more pronounced in epicotyls preliminarily starved in a phosphate solution for 24 hr. On the other hand, supplying shikimate to the epicotyls brought about no significant accumulation of quinic acid. Tracer studies with14C-shikimate have shown that, in the epicotyls, shikimic acid was rapidly metabolized and fairly high radioactivities were observed in the amino acid fraction. However, feeding of14C-shikimate together with unlabeled alicyclic acids resulted in a reduction of shikimate utilization. When3H-quinate was fed to the epicotyls, radioactivities were retained mostly in an acidic fraction, indicating the sluggish conversion of quinic acid. In starved epicotyls, however, nearly half of the absorbed radioactivity was consumed. In tracer experiments the conversion of quinate to shikimate was clearly observed, whereas the reverse reaction was not. From these findings the metabolic role of quinic acid in quinate-less pea seedlings is discussed.