Lipid-binding role of βII-spectrin ankyrin-binding domain

It is known that erythroid and non-erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially-expressed recombinant peptides representing beta II(brain)-spectrin's ankyrin-binding domain and its truncated mutants showed lipid-binding activity, although only those containing a full-length amino terminal fragment showed high to moderate affinity towards phospholipid mono- and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on beta I-spectrin's ankyrin-binding domain [Hryniewicz-Jankowska A, et al. Mapping of ankyrin-sensitive, PE/PC mono- and bilayer binding site in erytliroid beta-spectrin. Biochem J 2004;382:677-85]. Moreover, we tested also the effect of transient transfection of living cells of several cell-lines with vectors coding for GFP-conjugates including beta II and also beta I full-length ankyrin-binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full-length ankyrin-binding domain of beta II and beta I spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin-binding domain truncated at their N-terminal region. Our results therefore indicate the importance of N-terminal region for lipid-binding activity of the beta-spectrin ankyrin-binding domain and its substantial role in maintaining the spectrin-based skeleton distribution. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.