LABORATORY OF DEVELOPMENTAL REGULATION

Protein palmitoylation during embryogenesis

Covalent attachment of fatty acids is a feature of many proteins in eukaryotic cell. The two modifications involve acylation with myristate and palmitate. Myristate is usually linked through an amino-terminal glycine residue via an amide linkage which is relatively stable. Palmitate is usually linked through cysteine residue via a thioester linkage, and the post-translational process is referred to as protein palmitoylation. Protein palmitoylation is a dynamic modification. The turnover rate of bound palmitate through thioester linkages is faster than that of the modification with myristate through amide linkages. Most small GTP-binding, G proteins and G protein-coupled receptor proteins are known to be modified with palmitate via thioester linkages. Protein palmitoylation is thought to be important in the regulation of signal transduction. However the molecular mechanisms how protein palmitoylation regulate development is unknown.

We have previously found that protein palmitoylase is expressed in neural cells during mouse embryogenesis. In developing neurons growth associated protein (GAP)-43 and Go, which are palmitoylated proteins, are mainly concentrated in growth cones. Addition of an inhibitor of protein palmitoylase to the medium of cultured primary neural cells reduces the axonal growth of neurons. From these findings, we speculated that palmitoylation of the signaling proteins is critical for development of axons.

GAP-43 is a neuron-specific protein. Cys 3 and Cys 4 of GAP-43 are modified with palmitate in developing neurons. Transfection of GAP-43 cDNA into non-neural cells such as COS-7 cells is reported to cause the induction of filopodia-like formation. Since Cys residues are necessary for the induction of filopodia-like formation, protein palmitoylation is discussed to regulate the induction filopodia-like formation. Filopodial processes is thought be important for the development of neural growth cones. Therefore we focused to analyze the regulation mechanisms of the induction of filopodia-like formation in non-neural cells.

Transfection of protein palmitoylase cDNA into non-neural cells

We prepared the full-length protein palmitoylase cDNA for the expression in cultured cells. The cDNA contained a long open reading frame 2,504 amino acids. Protein palmitoylase and GAP-43 cDNAs were transfected into COS-7 and HeLa cells and the transient expression of these proteins were induced.

We confirmed that expression of protein palmitoylase reproducibly alters COS-7 morphology. Filopodia extention is observed in about 5% of control COS cells, but in 15% of protein palmitoylase-expressing COS cells under our conditions. To characterize the effect of protein palmitoylase on non-neural cells, we examined cellular morphology at various times after trypsinization and replating. Before contact with a substratum allowing adhesion, about 15% of control COS-7 cells exhibit filopodia. Transfection with protein palmitoylase increases this to 25%. The presence of filopodia is not significantly altered by varying the length of time to 120 minutes during which the cells are maintained in suspension after trypsinization. After contact with an adherent substratum the percentage of cells with filopodia rapidly declines in both the control and protein palmitoylase transfected cells. The rate of decline is approximately the same in both groups. The effect of protein palmitoylase is to alter the initial morphology after trypsinization, and not to alter the influence of the substratum on the cell shape.

Filopodia are exhibited by few protein palmitoylase expressing COS cells after 30 minutes of incubation with an appropriate substratum. However the shape of protein palmitoylase expressing cells can still be distinguished from control cells by another attribute, the degree of cell spreading. Protein palmitoylase expression appears to delay cell spreading. When control COS cells are plated on poly-L-lysine-coated glass, cell spreading occurs over 60 minutes. The time required to achieve the same degree of spreading is prolonged in the protein palmitoylase expressing cells, although the degree of spreading eventually reaches the same level. One day after plating, cell shape is indistinguishable between control and protein palmitoylase expressing cells.

Induction of filopodia-like formation in cultured cells by peptide transfection

We have established a method to chemically modify a biotinylated peptide composed of residues 1-15 of the GAP-43 N-terminal with fatty acids via thioester linkages. By using the method, we have prepared the peptides which are modified with myristate (C14:0), palmitate (C16:0), stearate (C18:0) and arachidate (C20:0) via thioester linkages. The method also modified the peptide with unsaturated fatty acid such as palmitoleate (C16:1). The modified peptides were separated by reverse phase HPLC and these peptides were eluted from the column depend on their chain length.

We are attempting to see the effect of filopodia-like formation in COS-7 cells by peptide transfection, which were chemically modified with fatty acids. Since the efficiency of the induction of filopodia-like formation by transfection with palmitoylated peptide is low, we are trying to elevate the efficiency.