The oleic acid complexes of proteolytic fragments of alpha-lactalbumin display apoptotic activity.
The complexes formed by partially folded human and bovine alpha-lactalbumin with oleic acid (OA) have been reported to display selective apoptotic activity against tumor cells. These complexes were named human (HAMLET) or bovine (BAMLET) alpha-lactalbumin made lethal to tumor cells. Here, we analyzed the OA complexes formed by fragments of bovine alpha-lactalbumin obtained by limited proteolysis of the protein. Specifically, the fragments investigated were 53-103 and the two-chain fragment species 1-40/53-123 and 1-40/104-123, these last being the N-terminal fragment 1-40 covalently linked via disulfide bridges to the C-terminal fragment 53-123 or 104-123. The OA complexes were obtained by mixing the fatty acid and the fragments in solution (10-fold and 15-fold molar excess of OA over protein fragment) or by chromatography of the fragments loaded onto an OA-conditioned anion exchange column and salt-induced elution of the OA complexes. Upon binding to OA, all fragments acquire an enhanced content of alpha-helical secondary structure. All OA complexes of the fragment species showed apoptotic activity for Jurkat tumor cells comparable to that displayed by the OA complex of the intact protein. We conclude that the entire sequence of the protein is not required to form an apoptotic OA complex, and we suggest that the apoptotic activity of a protein-OA complex does not imply specific binding of the protein.
Tolin
Serena
S
CRIBI Biotechnology Centre, University of Padua, Padua, Italy.
De Franceschi
Giorgia
G
Spolaore
Barbara
B
Frare
Erica
E
Canton
Marcella
M
Polverino de Laureto
Patrizia
P
Fontana
Angelo
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002417:Cattle; D006801:Humans; D066298:In Vitro Techniques; D019169:Jurkat Cells; D007768:Lactalbumin / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D046911:Macromolecular Substances; D009829:Oleic Acids / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013045:Species Specificity
C506422:HAMLET complex, human; D046911:Macromolecular Substances; D009829:Oleic Acids; D010446:Peptide Fragments; D007768:Lactalbumin