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. 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.


RESULTS
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
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.
Structural Glycobiology Section, CCR-Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, Maryland, United States of America.
Natalia
N
Mercer
Boopathy
B
Ramakrishnan
Elizabeth
E
Boeggeman
Pradman K
PK
Qasba
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D008958:Models, Molecular; D057054:Molecular Imaging; D008969:Molecular Sequence Data; D009829:Oleic Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity
D000970:Antineoplastic Agents; C506422:HAMLET complex, human; D009829:Oleic Acids; D001710:Biotin; D007768:Lactalbumin