Interaction between MARCKS and PSA

In a previous study, it has been demonstrated that MARCKS and PSA interact with each other (Bibhudetta Mishra, PhD thesis, Hamburg; Maren von der Ohen, PhD thesis, Hamburg). I have further characterized this interaction. Since MARCKS is an

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intracellular protein and PSA on NCAM is located extracellularly, it seems likely that the interaction between both molecules takes place from opposite sides of the plasma membrane.

The ED of MARCKS attaches to membranes via electrostatic interactions between its positively charged residues and the negatively charged phospholipids of the membrane bilayer. The phenylalanines of MARCKS ED can penetrate in addition through the polar head groups of the lipids of the bilayer into the hydrophopbic part of the plasma membrane, and thus, stabilizes the attachment of MARCKS to the plasma membrane (Victor et al., 1999; Qin et al., 1996). The helical and random coiled conformation of PSA (Brisson et al., 1992) facilitates its association with liposomal membranes and its penetration into lipid bilayers (Janas et al., 2000; 2001;

2010). Based on the similar properties of PSA and MARCKS to insert into the membrane, it could be inferred that PSA and MARCKS might thereby interact within the plasma membrane from opposite sites. The results from the cross-linking approach with photo-L-leucine and FRET analysis using different MARCKS-constructs and colominic acid/PSA or PSA-NCAM-Fc support this hypothesis.

Furthermore, application of the ED-peptide, to the opposite site colominic acid/PSA application destabilized the bilayer and allowed ions to flow through the bilayer and, therefore, to change the electrical properties of the membrane. Neither chondroitin sulfate nor the control peptide insert into the bilayer and thus do not change the properties of the membrane. This PSA-MARCKS interaction may form hydrophilic pores in the lipid bilayer, which would allow ions to pass the membrane. Since PSA is able to influence the long term potentiation in the brain (Becker et al., 1996; Muller et al., 1996), this observation suggests a novel mechanism of how PSA could modulate the membrane potential to influence cellular processes involved in long term potentiation.

The PSA-enhanced neurite outgrowth in hippocampal neurons was triggered upon colominic acid/PSA incubation (Mehanna et al., 2010; Muller et al., 1994). This PSA-enhanced neurite outgrowth was inhibited by transfection or by extracellular addition of the MARCKS ED peptide, but not by application of the MARCKS control peptide.

Moreover, levels of phosphorylated MARCKS in hippocampal neurons were

Discussion

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decreased after PSA treatment. Based on this observation, one could speculate that the additionally added colominic acid might recruit more MARCKS at the plasma membrane; in order to prevent the phosphorylation of MARCKS ED. Phosphorylated MARCKS may further trigger a signal cascade, which inhibits neurite outgrowth.

Application of additional PSA would then lead to an inhibition of this signal cascade and, consequently, to an enhanced neurite outgrowth. The MARCKS ED peptide may disturb the PSA-MARCKS interaction, which might lead to an increased phosphorylation of MARCKS and, thereby, stimulate the signal cascade, which thereafter inhibits neurite outgrowth.

NCAM-deficient neurons showed a decreased neurite outgrowth after heparin treatment and an enhanced neurite outgrowth after chondroitin sulfate treatment. In contrast, heparin and chondroitin sulfate had no effect on neurite outgrowth of wild-type neurons. In its Ig1 and Ig2 domains NCAM contains one heparin binding motif to which both heparin and chondroitin sulfate can bind (Cole et al., 1989, 1986a, b).

One possible explanation, that an effect of heparin and chondroitin sulfate in neurite outgrowth of NCAM-deficient, but not wild-type hippocampal neurons, was observed would be that heparin and chondroitin sulfate could interact with the heparin binding motif of NCAM in wild-type neurons. This might lead to a neutralization of the heparin and chondroitin sulfate effect on neurite outgrowth of wild-type, but not NCAM-deficient hippocampal neurons.

It was reported that the MARCKS ED peptide has an enhancing effect on hippocampal neuritogenesis (Theis et al., 2013). One can assume that the inhibitory effect of the ED peptide on NCAM-mediated neurite outgrowth could be explained by a side effect of this ED peptide, and not disturb a possible PSA-MARCKS interaction.

Since the phenylalanine residues in the ED of MARCKS show a high affinity of binding to PIP2, and thus, regulate the availability of PIP2 by sequestration of PIP2

(Gambhir et al., 2004; Laux et al., 2000), which has been implicated in signal transduction (Kwiatkowska et al., 2010), it seems conceivable, that this sequestrated of PIP2 leads to an increased total neurite length of hippocampal neurons. In contrast, the additional treatment of hippocampal neurons with PSA would lead to inhibition of neuritogenesis. The results from this study are contrary to this

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assumption. Here, the ED peptide had no stimulating effect on neuritogenesis in hippocampal neurons from NCAM deficient mice, which also express PIP2, but do not PSA. Additonally, the ED peptide was applied to the neurons extracellulary which did not allow the peptide to sequestrate PIP2, because PIP2 is abundantly present in the inner layer of the plasma membrane. Since the ED peptide stimulates neurite outgrowth only in the presence of endogenous PSA, it seems plausible that the ED peptide disturbs the interaction between PSA and MARCKS, and thus, might provide PSA which is accessible for other interaction partners, such as histone H1 or FGF.

This interaction of PSA with histone H1 or FGF would lead to an increase of the neurite outgrowth of hippocampal neurons. My assumptions seem controversial.

However, endogenous PSA may recruit much less MARCKS at the plasma membrane which prevent MARCKS to be phosporylated, then additional applied colominic acid/PSA to the neurons. The stimulation of neurite outgrwoth triggered by other interaction partners then MARCKS, such as histone H1 or FGF, seems to be stronger then the inhibition of neurite outgrowth by additionally phosphorylated MARCKS.

Literature

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