Applications of site-specific labeling to study HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.
Applications of site-specific labeling to study HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.
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DOI：
10.1371/journal.pone.0026093
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Qasba PK
中科院分区：
文献类型：
--
作者：
Mercer N;Ramakrishnan B;Boeggeman E;Qasba PK
α-乳白蛋白（α-LA）是一种钙结合的乳腺特异性蛋白质，存在于乳汁中。这种蛋白质是β 1，4-半乳糖基转移酶的调节剂，将其受体特异性从N-乙酰葡糖胺改变为葡萄糖，以产生乳糖，牛奶的主要碳水化合物。当从α-LA中除去钙时，它采用熔融球的形式，有趣的是，这种形式与油酸（OA）复合时获得了杀肿瘤活性。这种由人α-LA（hLA）制成的复合物被称为哈姆雷特（对肿瘤细胞致命的人A-乳白蛋白），并且其杀肿瘤活性已被充分确立。在目前的工作中，我们已经使用了位点特异性标记，在我们的实验室以前开发的技术，标记哈姆雷特与生物素，或荧光探针共聚焦显微镜研究。除全长hLA外，本研究还包括单独的hLA α结构域（αD-hLA）。我们已经用17个氨基酸的C-末端延伸（hLA-ext和αD-hLA-ext）改造了这些蛋白质。使用多肽-α-N-乙酰半乳糖胺转移酶II（ppGalNAc-T2）将该延伸中的单个Thr残基用2-丙酮基-半乳糖（C2-酮基-半乳糖）糖基化，并进一步与荧光探针或生物素分子的氨氧基衍生物缀合。我们发现，hLA和αD-hLA蛋白的熔融球形式，无论有或没有C-末端延伸，无论有或没有缀合的荧光探针或生物素分子，都容易与OA形成复合物，并表现出与用全长hLA蛋白制备的哈姆雷特相似的杀肿瘤活性。荧光探针标记的样品的共聚焦显微镜研究表明，这些蛋白质被内化到细胞中，甚至在细胞核中发现，只有当它们与OA复合。与单个生物素分子缀合的哈姆雷特将是鉴定与其参与杀肿瘤活性的细胞组分的有用工具。
Alpha-lactalbumin (α-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of β1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milk's main carbohydrate. When calcium is removed from α-LA, it adopts a molten globule form, and this form, interestingly, when complexed with oleic acid (OA) acquires tumoricidal activity. Such a complex made from human α-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established. In the present work, we have used site-specific labeling, a technique previously developed in our laboratory, to label HAMLET with biotin, or a fluoroprobe for confocal microscopy studies. In addition to full length hLA, the α-domain of hLA (αD-hLA) alone is also included in the present study. We have engineered these proteins with a 17–amino acid C-terminal extension (hLA-ext and αD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose) using polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules. We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity.
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DOI：
10.1016/j.bbrc.2008.08.127
发表时间：
2008-11-07
影响因子：
3.1
作者：
Kamijima, Tatsuro;Ohmura, Ayaka;Demura, Makoto
通讯作者：
Demura, Makoto
影响因子：
6.4
作者：
Aits, Sonja;Gustafsson, Lotta;Svanborg, Catharina
通讯作者：
Svanborg, Catharina
影响因子：
3.7
作者：
Mossberg AK;Puchades M;Halskau Ø;Baumann A;Lanekoff I;Chao Y;Martinez A;Svanborg C;Karlsson R
通讯作者：
Karlsson R
DOI：
10.1007/978-0-387-74087-4_8
发表时间：
2008-01-01
期刊：
BIOACTIVE COMPONENTS OF MILK
影响因子：
--
作者：
Hallgren, Oskar;Aits, Sonja;Svanborg, Catharina
通讯作者：
Svanborg, Catharina
影响因子：
64.8
作者：
QASBA, PK;SAFAYA, SK
通讯作者：
SAFAYA, SK