title: Datura stramonium agglutinin: cloning, molecular characterization and recombinant production in Arabidopsis thaliana.
abstract: Datura stramonium seeds contain at least three chitin-binding isolectins [termed Datura stramonium agglutinin (DSA)] as homo- or heterodimers of A and B subunits. We isolated a cDNA encoding isolectin B (DSA-B) from an immature fruit cDNA library; this contained an open reading frame encoding 279 deduced amino acids, which was confirmed by partial sequencing of the native DSA-B peptide. The sequence consisted of: (i) a cysteine (Cys)-rich carbohydrate-binding domain composed of four conserved chitin-binding domains and (ii) an extensin-like domain of 37 residues containing four SerPro4-6 motifs that was inserted between the second and third chitin-binding domains (CBDs). Although each chitin-binding domain contained eight conserved Cys residues, only the second chitin-binding domain contained an extra Cys residue, which may participate in dimerization through inter-disulfide bridge formation. Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, the molecular mass of homodimeric lectin composed of two B-subunits was determined as 68,821 Da. The molecular mass of the S-pyridilethylated B-subunit were found to be 37,748 Da and that of the de-glycosylated form was 26,491 Da, which correlated with the molecular weight estimated from the deduced sequence. Transgenic Arabidopsis plants overexpressing the dsa-b demonstrated hemagglutinating activity. Recombinant DSA-B was produced as a homodimeric glycoprotein with a similar molecular mass to that of the native form. Moreover, the N-terminus of the purified recombinant DSA-B protein was identical to that of the native DSA-B, confirming that the cloned cDNA encoded DSA-B.
authors.affiliation: Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido 099-2493, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Nishimoto
authors.affiliation: Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido 099-2493, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Tanaka
authors.affiliation: Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido 099-2493, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Murakami
authors.affiliation: Department of Applied Biology and Chemistry, Tokyo University of Agriculture, Setagaya, Tokyo 156-8502, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakashita
authors.affiliation: Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido 099-2493, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Sakamoto
authors.affiliation: Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido 099-2493, Japan s-oguri@bioindustry.nodai.ac.jp.
authors.forename: Suguru
authors.initials: S
authors.lastname: Oguri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D013286:Datura stramonium / Q000235:genetics*; D004912:Erythrocytes / Q000187:drug effects; D006031:Glycosylation; D006384:Hemagglutination / Q000187:drug effects; D006388:Hemagglutinins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
chemical_list: D006388:Hemagglutinins; D037121:Plant Lectins; D011994:Recombinant Proteins
keywords: DSA; Datura stramonium; Solanaceae; chitin-binding; recombinant lectin