Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid 英文参考文献.docVIP

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Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid 英文参考文献.doc

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Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid 英文参考文献

ApplicationsofSite-SpecificLabelingtoStudyHAMLET, aTumoricidalComplexofa-LactalbuminandOleicAcid NataliaMercer1,BoopathyRamakrishnan1,2,ElizabethBoeggeman1,2,PradmanK.Qasba1* 1StructuralGlycobiologySection,CCR-NanobiologyProgram,CenterforCancerResearch,NCI-Frederick,Frederick,Maryland,UnitedStatesofAmerica,2BasicScience Program,SAIC-Frederick,Inc.,NCI-Frederick,Frederick,Maryland,UnitedStatesofAmerica Abstract Background:Alpha-lactalbumin(a-LA)isacalcium-boundmammarygland-specificproteinthatisfoundinmilk.Thisproteinisa modulator of b1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to producelactose,milk’smaincarbohydrate.Whencalciumisremovedfroma-LA,itadoptsamoltenglobuleform,andthisform, interestingly,whencomplexedwitholeicacid(OA)acquirestumoricidalactivity.Suchacomplexmadefromhumana-LA(hLA)is knownasHAMLET(HumanA-lactalbuminMadeLethaltoTumorcells),anditstumoricidalactivityhasbeenwellestablished. Methodology/Principal Findings: In the present work, we have used site-specific labeling, a technique previously developedinourlaboratory,tolabelHAMLETwithbiotin,orafluoroprobeforconfocalmicroscopystudies.Inadditionto full length hLA, the a-domain of hLA (aD-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 aD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose) using polypeptide-a-N-acetylgalactosaminyltransferase II (ppGalNAc-T2)andfurtherconjugatedwithaminooxy-derivativesoffluoroprobeorbiotinmolecules. Conclusions/Significance:WefoundthatthemoltenglobuleformofhLAandaD-hLAproteins,withorwithoutC-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibitstumoricidal activity similar toHAMLET made withfull-length hLA protein.The confocal microscopy studies with fluoroprobe-labeledsamplesshowthatth