Nishimoto Keisuke, Tanaka Kaori, Murakami Takahiro, Nakashita Hideo, Sakamoto Hikaru, Oguri Suguru
Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido 099-2493, Japan.
Department of Applied Biology and Chemistry, Tokyo University of Agriculture, Setagaya, Tokyo 156-8502, Japan.
Glycobiology. 2015 Feb;25(2):157-69. doi: 10.1093/glycob/cwu098. Epub 2014 Sep 22.
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.
曼陀罗种子含有至少三种几丁质结合异凝集素[称为曼陀罗凝集素（DSA）]，它们是A和B亚基的同二聚体或异二聚体。我们从未成熟果实cDNA文库中分离出编码异凝集素B（DSA-B）的cDNA；它包含一个编码279个推导氨基酸的开放阅读框，这通过对天然DSA-B肽的部分测序得到证实。该序列由：（i）一个富含半胱氨酸（Cys）的碳水化合物结合结构域组成，该结构域由四个保守的几丁质结合结构域和（ii）一个37个残基的伸展蛋白样结构域组成，该结构域包含四个SerPro4-6基序，插入在第二个和第三个几丁质结合结构域（CBD）之间。虽然每个几丁质结合结构域都含有八个保守的Cys残基，但只有第二个几丁质结合结构域含有一个额外的Cys残基，它可能通过形成链间二硫键参与二聚化。使用基质辅助激光解吸/电离飞行时间质谱法，由两个B亚基组成的同二聚体凝集素的分子量测定为68,821 Da。发现S-吡啶乙基化B亚基的分子量为37,748 Da，去糖基化形式的分子量为26,491 Da，这与从推导序列估计的分子量相关。过表达dsa-b的转基因拟南芥植物表现出血凝活性。重组DSA-B作为同二聚体糖蛋白产生，其分子量与天然形式相似。此外，纯化的重组DSA-B蛋白的N末端与天然DSA-B的N末端相同，证实克隆的cDNA编码DSA-B。
