Yang Hsiuchin, Johnson Paul Micah, Ko Ko-Chun, Kamio Michiya, Germann Markus W, Derby Charles D, Tai Phang C
Department of Biology, Georgia State University, Atlanta, GA 30302-4010, USA.
J Exp Biol. 2005 Sep;208(Pt 18):3609-22. doi: 10.1242/jeb.01795.
A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica was cloned and sequenced, and is the first sea hare antimicrobial protein to be functionally expressed in E. coli. Sequence analysis suggested that this protein is a flavin-containing l-amino acid oxidase (LAAO), with one predicted potential glycosylation site, although the glycosylation could not be experimentally confirmed. This protein, which we call ;escapin', has high sequence similarity to several other gastropod proteins. Escapin was verified by NMR, mass spectroscopy and HPLC to have FAD as its flavin cofactor. Escapin's antimicrobial effects, bacteriostasis and bactericidal, were determined using a combination of two assays: (1) incubation of bacteria on solid media followed by assessment of inhibition by direct observation of zones of inhibition or by turbidity measurements; and (2) incubation of bacteria in liquid media followed by counting viable colonies after growing on agar plates. Native escapin inhibited the growth of Gram-positive and Gram-negative bacteria, including marine bacteria (Vibrio harveyii and Staphylococcus aureus) and pathogenic bacteria (Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa). Escapin also inhibited the growth of yeast and fungi, with different efficacies. Escapin's antimicrobial activity was concentration dependent and did not decrease when stored for more than 5 months at room temperature. Escapin was bacteriostatic and not bactericidal in minimal media (e.g. salt media) with glucose, yeast extract, and a mixture of 20 amino acids each at 50 micromol l(-1), but was bactericidal in media enriched with Tryptone Peptone. Escapin was also strongly bactericidal in media with l-lysine at concentrations as low as 3 mmol l(-1) and slightly bactericidal in 50 mmol l(-1) l-arginine, but not in most other amino acids even at 50 mmol l(-1). Escapin had high oxidase activity (producing hydrogen peroxide) with either l-arginine or l-lysine as a substrate and little to no oxidase activity with other l-amino acids. Hydrogen peroxide alone (without escapin or amino acids) was strongly bacteriostatic but poorly bactericidal, similar in this respect to l-arginine but different from l-lysine in the presence of escapin. Together these results suggest that there are multiple mechanisms to escapin's antimicrobial effects, with bacteriostasis resulting largely or entirely from the effects of hydrogen peroxide produced by escapin's LAAO activity, but bactericidal effects resulting from lysine-dependent mechanisms not directly involving hydrogen peroxide. Recombinant escapin expressed in bacteria was also active against Gram-positive and Gram-negative bacteria, suggesting that glycosylation is not essential for antimicrobial activity.
从海兔加州海兔防御性紫色墨汁分泌物中分离出一种60 kDa的单体蛋白，对其进行了克隆和测序，它是首个在大肠杆菌中实现功能表达的海兔抗菌蛋白。序列分析表明，该蛋白是一种含黄素的L-氨基酸氧化酶（LAAO），有一个预测的潜在糖基化位点，不过糖基化无法通过实验证实。我们将这种蛋白称为“逃逸素”，它与其他几种腹足纲动物蛋白具有高度的序列相似性。通过核磁共振、质谱和高效液相色谱验证，逃逸素以黄素腺嘌呤二核苷酸（FAD）作为其黄素辅因子。使用两种检测方法相结合来确定逃逸素的抗菌效果，即抑菌和杀菌作用：（1）将细菌接种在固体培养基上，然后通过直接观察抑菌圈或测量浊度来评估抑制作用；（2）将细菌接种在液体培养基中，然后在琼脂平板上培养后计算活菌菌落数。天然的逃逸素能抑制革兰氏阳性菌和革兰氏阴性菌的生长，包括海洋细菌（哈维氏弧菌和金黄色葡萄球菌）和病原菌（金黄色葡萄球菌、化脓性链球菌和铜绿假单胞菌）。逃逸素也能抑制酵母和真菌的生长，但其效果不同。逃逸素的抗菌活性具有浓度依赖性，在室温下储存5个多月后活性并未降低。在含有葡萄糖、酵母提取物和20种各50 μmol l⁻¹氨基酸混合物的基本培养基（如盐培养基）中，逃逸素具有抑菌作用但无杀菌作用，但在富含胰蛋白胨的培养基中具有杀菌作用。在含有低至3 mmol l⁻¹ L-赖氨酸的培养基中，逃逸素也具有很强的杀菌作用，在50 mmol l⁻¹ L-精氨酸中具有轻微杀菌作用，但在大多数其他氨基酸中即使浓度达到50 mmol l⁻¹也无杀菌作用。以L-精氨酸或L-赖氨酸为底物时，逃逸素具有较高的氧化酶活性（产生过氧化氢），而以其他L-氨基酸为底物时氧化酶活性很低或几乎没有。单独的过氧化氢（无逃逸素或氨基酸）具有很强的抑菌作用但杀菌作用较弱，在这方面与L-精氨酸相似，但在有逃逸素存在时与L-赖氨酸不同。这些结果共同表明，逃逸素的抗菌作用有多种机制，抑菌作用主要或完全是由逃逸素的LAAO活性产生的过氧化氢的作用导致的，但杀菌作用是由不直接涉及过氧化氢的赖氨酸依赖性机制导致的。在细菌中表达的重组逃逸素对革兰氏阳性菌和革兰氏阴性菌也有活性，这表明糖基化对于抗菌活性并非必不可少。
