Witold Diakowski

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC). In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site. In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.

Lipid raft-dependent endocytosis of close homolog of adhesion molecule L1 (CHL1) promotes neuritogenesis.

Background: Cell adhesion molecule CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. Results: Ligand-induced clustering of CHL1 at the cell surface induces lipid raft-dependent endocytosis of CHL1 and neuritogenesis. Conclusion: Ligand-induced remodeling of CHL1 adhesion promotes neurite outgrowth. Significance: High levels of CHL1 adhesion and inhibition of CHL1 endocytosis can interfere with neuronal development and/or regeneration. CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. We report here that neuritogenesis-promoting ligand-dependent cell surface clustering of CHL1 induces palmitoylation and lipid raft-dependent endocytosis of CHL1. We identify βII spectrin as a binding partner of CHL1, and we show that partial disruption of the complex between CHL1 and βII spectrin accompanies CHL1 endocytosis. Inhibition of the association of CHL1 with lipid rafts by pharmacological disruption of lipid rafts or by mutation of cysteine 1102 within the intracellular domain of CHL1 reduces endocytosis of CHL1. Endocytosis of CHL1 is also reduced by nifedipine, an inhibitor of the L-type voltage-dependent Ca2+ channels. CHL1-dependent neurite outgrowth is reduced by inhibitors of lipid raft assembly, inhibitors of voltage-dependent Ca2+ channels, and overexpression of CHL1 with mutated cysteine Cys-1102. Our results suggest that ligand-induced and lipid raft-dependent regulation of CHL1 adhesion via Ca2+-dependent remodeling of the CHL1-βII spectrin complex and CHL1 endocytosis are required for CHL1-dependent neurite outgrowth.

Structural insight into an ankyrin-sensitive lipid-binding site of erythroid β-spectrin

It was recently shown that the region within β-spectrin responsible for interactions with ankyrin includes a lipid-binding site which displayed sensitivity to inhibition by ankyrin. We studied its structure by constructing a series of single and double spin-labeled β-spectrin-derived peptides and analyzing their spin-spin distances via electron paramagnetic resonance spectroscopy and the Fourier deconvolution method. The results indicate that the whole ankyrin-sensitive lipid-binding site of β-spectrin exhibits a helical conformation revealing a distinct 310-helix contribution at its N-terminus. The start of the helix was located five residues upstream along the sequence compared to the theoretical predictions. A model based on the obtained data provides direct evidence that the examined lipid-binding site is a highly amphipathic helix, which is correlated with the specific conformation of its N-terminal fragment.

Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.

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 βII(brain)‐spectrins 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 βI‐spectrins 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 βII and also βI 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 βII and βI 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 β‐spectrin ankyrin‐binding domain and its substantial role in maintaining the spectrin‐based skeleton distribution.

Occurrence of lipid receptors inferred from brain and erythrocyte spectrins binding NaOH-extracted and protease-treated neuronal and erythrocyte membranes

It was previously shown in model systems that brain spectrin binds membrane phospholipids. In the present study, we analysed binding of isolated brain spectrin and red blood cell spectrin to red blood or neuronal membranes which had been treated as follows: (1). extracted with low ionic-strength solution, (2). the above membranes extracted with 0.1 M NaOH, and (3). membranes treated as above, followed by protease treatment and re-extraction with 0.1 M NaOH. It was found that isolated, NaOH-extracted, protease-treated neuronal and red blood cell membranes bind brain and red blood cell spectrin with moderate affinities similar to those obtained in model phospholipid membrane-spectrin interaction experiments. Moreover, this binding was competitively inhibited by liposomes prepared from membrane lipids. The presented results indicate the occurrence of receptor sites for spectrins that are extraction- and protease-resistant, therefore most probably of lipidic nature, in native membranes.

Mitoxantrone changes spectrin-aminophospholipid interactions.

Understanding drug-membrane and drug-membrane protein interactions would be a crucial step towards understanding the action and biological properties of anthracyclines, as the cell membrane with its integral and peripheral proteins is the first barrier encountered by these drugs. In this paper, we briefly describe mitoxantrone-monolayer and mitoxantrone-bilayer interactions, focusing on the effect of mitoxantrone on the interactions between erythroid or nonerythroid spectrin with phosphatidylethanolamine-enriched mono- and bilayers. We found that mitoxantrone markedly modifies the interaction of erythroid and nonerythroid spectrins with phosphatidylethanolamine/phosphatitydcholine (PE/PC) monolayers. The change in Δπ induced by spectrins is several-fold larger in the presence of 72 nM mitoxantrone than in its absence: spectrin/mitoxantrone complexes induced a strong compression of the monolayer. Spin-labelling experiments showed that spectrin/mitoxantrone complexes caused significant changes in the order parameter measured using a 5′-doxyl stearate probe in the bilayer, but they practically did not affect the mobility of 16′-doxyl stearate. These results indicate close-to-surface interactions/penetrations without significant effect on the mid-region of the hydrophobic core of the bilayer. The obtained apparent equilibrium dissociation constants indicated relatively similar mitoxantrone-phospholipid and mitoxantrone-spectrin (erythroid and nonerythroid) binding affinities. These results might in part, explain the effect of mitoxantrone on spectrin distribution in the living cells.

Chapter Four Interactions of Erythroid and Nonerythroid Spectrins and Other Membrane-Skeletal Proteins with Lipid Mono- and Bilayers

Abstract The object of the chapter is to review the studies on the interactions of erythroid and nonerythroid spectrins and other membrane-skeletal proteins with lipids in model membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Important issue is the mechanism(s) involved in these interactions.