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Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid

BACKGROUND: 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. W...

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Autores principales: Mercer, Natalia, Ramakrishnan, Boopathy, Boeggeman, Elizabeth, Qasba, Pradman K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189925/
https://www.ncbi.nlm.nih.gov/pubmed/22016817
http://dx.doi.org/10.1371/journal.pone.0026093
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author Mercer, Natalia
Ramakrishnan, Boopathy
Boeggeman, Elizabeth
Qasba, Pradman K.
author_facet Mercer, Natalia
Ramakrishnan, Boopathy
Boeggeman, Elizabeth
Qasba, Pradman K.
author_sort Mercer, Natalia
collection PubMed
description BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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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spelling pubmed-31899252011-10-20 Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid Mercer, Natalia Ramakrishnan, Boopathy Boeggeman, Elizabeth Qasba, Pradman K. PLoS One Research Article BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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. Public Library of Science 2011-10-10 /pmc/articles/PMC3189925/ /pubmed/22016817 http://dx.doi.org/10.1371/journal.pone.0026093 Text en This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
Mercer, Natalia
Ramakrishnan, Boopathy
Boeggeman, Elizabeth
Qasba, Pradman K.
Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid
title Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid
title_full Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid
title_fullStr Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid
title_full_unstemmed Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid
title_short Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid
title_sort applications of site-specific labeling to study hamlet, a tumoricidal complex of α-lactalbumin and oleic acid
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189925/
https://www.ncbi.nlm.nih.gov/pubmed/22016817
http://dx.doi.org/10.1371/journal.pone.0026093
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