East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China; Key Laboratory of East China Sea and Oceanic Fishery Resources Exploitation, Ministry of Agriculture, Shanghai 200090, China.
School of Life Sciences, Shandong University, Jinan, Shandong 250100, China.
Fish Shellfish Immunol. 2014 Jan;36(1):172-80. doi: 10.1016/j.fsi.2013.10.023. Epub 2013 Nov 15.
Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with binding and neutralizing activities to lipopolysaccharide (LPS) in crustaceans. This study identified and characterized a novel ALF homolog (SpALF4) from the mud crab Scylla paramamosain. The complete cDNA of SpALF4 had 756 bp with a 381 bp open reading frame encoding a protein with 126 aa. The deduced protein contained a signal peptide and a LPS-binding domain. SpALF4 shared the highest identity with PtALF5 at amino acid level but exhibited low similarity with most of other crustacean ALFs. Furthermore, different from the previously identified three SpALF homologs and most of other ALFs, SpALF4 had a low isoelectric point (pI) for the mature peptide and the LPS-binding domain with the values of 6.93 and 6.74, respectively. These results indicate that SpALF4 may be a unique ALF homolog with special biological function in the mud crab. Similar to the spatial structure of ALFPm3, SpALF4 contains three α-helices packed against a four-strand β-sheet, and an amphipathic loop formed by a disulphide bond between two conserved cysteine residues in LPS-binding domain. SpALF4, mainly distributed in hemocytes, could be upregulated by Vibrio harveyi, Staphylococcus aureus, or white spot syndrome virus. Recombinant SpALF4 could inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio anguillarum, Vibrio alginolyticus, Aeromonas hydrophila, Pseudomonas putida), Gram-positive bacteria (S. aureus and Bacillus megaterium), and a fungus Candida albicans to varying degrees. Further study showed that it could also bind to all the aforementioned microorganisms except S. aureus. These results demonstrate that SpALF4 is a unique ALF homolog with potent antimicrobial activity against bacteria and fungi. This characteristic suggests SpALF4 plays an essential function in immune defense against pathogen invasion in mud crab.
抗脂多糖因子（ALFs）是具有结合和中和甲壳动物脂多糖（LPS）活性的抗菌肽。本研究从泥蟹锯缘青蟹中鉴定和表征了一种新型的 ALF 同源物（SpALF4）。SpALF4 的完整 cDNA 长 756bp，开放阅读框长 381bp，编码 126 个氨基酸的蛋白质。推导的蛋白质包含一个信号肽和一个 LPS 结合结构域。SpALF4 在氨基酸水平上与 PtALF5 的同源性最高，但与大多数其他甲壳动物的 ALFs 的相似性较低。此外，与先前鉴定的三个 SpALF 同源物和大多数其他 ALFs 不同，SpALF4 的成熟肽的等电点（pI）和 LPS 结合结构域的 pI 较低，分别为 6.93 和 6.74。这些结果表明 SpALF4 可能是一种具有特殊生物学功能的独特 ALF 同源物。与 ALFPm3 的空间结构相似，SpALF4 包含三个α-螺旋，排列在一个四链β-折叠上，以及一个由两个保守半胱氨酸残基之间的二硫键形成的两亲性环，位于 LPS 结合结构域中。SpALF4 主要分布在血细胞中，可被哈维弧菌、金黄色葡萄球菌或白斑综合征病毒上调。重组 SpALF4 可不同程度地抑制革兰氏阴性菌（哈维弧菌、鳗弧菌、溶藻弧菌、嗜水气单胞菌、恶臭假单胞菌）、革兰氏阳性菌（金黄色葡萄球菌和巨大芽孢杆菌）和真菌白色念珠菌的生长。进一步的研究表明，它还可以与除金黄色葡萄球菌以外的所有上述微生物结合。这些结果表明 SpALF4 是一种具有抗细菌和真菌活性的独特 ALF 同源物。这一特性表明 SpALF4 在泥蟹抵御病原体入侵的免疫防御中发挥着重要作用。
