Imjongjirak Chanprapa, Amparyup Piti, Tassanakajon Anchalee, Sittipraneed Siriporn
Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Mol Immunol. 2007 May;44(12):3195-203. doi: 10.1016/j.molimm.2007.01.028. Epub 2007 Mar 26.
Antilipopolysaccharide factors (ALFs) are small basic proteins that can bind and neutralize lipopolysaccharide (LPS) and have broad spectrum antimicrobial activities. In this study, we describe the isolation of the full-length cDNA encoding for ALF peptide (ALFSp) of mud crab, Scylla paramamosain by sequencing a hemocyte cDNA library and using the rapid amplification cDNA end (RACE) method. A full-length ALFSp cDNA of 614 bp contains an open reading frame (ORF) of 372 bp, encoding 123 amino acid protein with 26 residues signal sequence. The calculated molecular mass of the mature protein is 11.18 kDa. The highly two conserve cysteine residues and putative LPS binding domain were observed in ALFSp peptide. Comparison of amino acid sequences revealed that ALFSp shared high identity with other known ALFs and had an overall similarity of 65, 64, 63, 61 and 59% to those of Fenneropenaeus chinensis, Litopenaeus vannamei, Marsupenaeus japonicus, Limulus polyphemus, and Tachypleus tridentatus, respectively. A neighbour-joining tree showed a clear differentiation of each species and also indicated that ALF from S. paramamosain, Carcinus maenas and Callinectes sapidus are closely related phylogenetically. The genomic DNA sequence of ALFSp gene consists of 1075 bp containing three exons and two introns. Tissue distribution analysis revealed that ALFSp was abundantly expressed in hemocytes, intestine, and muscle but not in eyestalk. The synthetic ALFSp peptide containing putative LPS binding domain revealed a strong antimicrobial activity against several bacteria especially on the growth of Gram-positive bacteria, Micrococcus luteus and Gram-negative bacteria, Vibrio harveyi suggested that ALFSp could play an essential role in defense mechanism in S. paramamosain.
抗脂多糖因子（ALFs）是一类小分子碱性蛋白，能够结合并中和脂多糖（LPS），具有广谱抗菌活性。在本研究中，我们通过对锯缘青蟹血细胞cDNA文库进行测序，并采用快速扩增cDNA末端（RACE）方法，描述了编码锯缘青蟹ALF肽（ALFSp）的全长cDNA的分离过程。一个614 bp的全长ALFSp cDNA包含一个372 bp的开放阅读框（ORF），编码一个由123个氨基酸组成的蛋白质，带有26个残基的信号序列。成熟蛋白的计算分子量为11.18 kDa。在ALFSp肽中观察到两个高度保守的半胱氨酸残基和假定的LPS结合结构域。氨基酸序列比较显示，ALFSp与其他已知的ALFs具有高度同源性，与中国对虾、凡纳滨对虾、日本囊对虾、美洲鲎和三刺鲎的ALFs的总体相似性分别为65%、64%、63%、61%和59%。邻接法构建的系统发育树显示了每个物种的明显分化，也表明锯缘青蟹、欧洲绿蟹和美味优游蟹的ALF在系统发育上密切相关。ALFSp基因的基因组DNA序列由1075 bp组成，包含三个外显子和两个内含子。组织分布分析表明，ALFSp在血细胞、肠道和肌肉中大量表达，但在眼柄中不表达。含有假定LPS结合结构域的合成ALFSp肽对几种细菌具有很强的抗菌活性，特别是对革兰氏阳性菌藤黄微球菌和革兰氏阴性菌哈维氏弧菌的生长有抑制作用，这表明ALFSp可能在锯缘青蟹的防御机制中发挥重要作用。
