Lipid-binding role of betaII-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 betaII(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 betaI-spectrin's ankyrin-binding domain [Hryniewicz-Jankowska A, et al. Mapping of ankyrin-sensitive, PE/PC mono- and bilayer binding site in erythroid 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 betaII and also betaI 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 betaII and betaI 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.
Bok
Ewa
E
Institute of Biochemistry and Molecular Biology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-149 Wrocław, Poland.
Plazuk
Ewa
E
Hryniewicz-Jankowska
Anita
A
Chorzalska
Anna
A
Szmaj
Agnieszka
A
Dubielecka
Patrycja M
PM
Stebelska
Katarzyna
K
Diakowski
Witold
W
Lisowski
Marek
M
Langner
Marek
M
Sikorski
Aleksander F
AF
D000465:Algorithms; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008546:Melanoma, Experimental / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013049:Spectrin
D017487:Ankyrins; D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D008081:Liposomes; D008563:Membrane Lipids; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D013049:Spectrin