Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes

Abstract

The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P₂ (PIP₂) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.

Keywords: Chemical cytometry; Fluorescent reporter; Lipid enzyme; Phosphatidylinositol.

Figure 1. Biophysical and kinetic analysis of fluorescent PtdIns(4,5)P₂ analogs

A. Chemical structures of PtdIns(4,5)P₂ derivatives. B. Kinetic analysis of PI3K reaction with fluorescent PtdIns(4,5)P2 derivatives. Soluble PI3K assays were used to calculate the kinetic constant of the various fluorescent derivatives. Kinetic constants for the C6 reporter were not obtained due to the lack of production of the corresponding PtdIns(3,4,5)P₃. C. Fluorescent PtdIns(4,5)P₂ Substrate Competition. Soluble PI3K assay conditions were used to analyze the effects of multiple reporters in solution. Production of PtdIns(3,4,5)P₃ was monitored from reactions that contained a single fluorescent PtdIns(4,5)P₂ derivative (20 μM), short chain C9 or long chain C15, as well as production of each product in reactions that contained both reporter in equimolar (20 μM) concentrations. D. Representative chromatogram of separation of PIP₂-C9, PIP₂-C15, PIP₃-C9 and PIP₃-C15 by capillary electrophoresis (CE).

Figure 2. Competition with endogenous PtdIns(4,5)P₂ in the presence of liposome

A. An in vitro flotation assay was utilized to analyze the effects of substrate hydrophobicity on membrane association using liposomes formulated to mimic the lipid content of the plasma membrane. Fluorescence was analyzed before and after centrifugation and the various layers analyzed by capillary electrophoresis (CE). B. Liposomes were formulated to contain 10 μM total PtdIns(4,5)P₂ using fluorescent C15 PtdIns(4,5)P₂ derivative or endogenous PtdIns(4,5,)P₂ and 10 μM of PS as a carrier lipid. Production of PtdIns(3,4,5)P₃ was monitored to analyze the ability of the reporter to be metabolized in the presence of endogenous substrate.

Figure 3. Biophysical and kinetic analysis of fluorescent PtdIns analogs

A. Chemical structures of PtdIns derivatives. B. Kinetic analysis of PI4K reaction with fluorescent PtdIns derivatives. C. Representative images of fluorescent PtdIns derivatives interacting with liposome. The presentation of the fluorescent lipids in either liposome or aqueous layer was quantified by CE.

Scheme 1

Synthesis of PtdIns(4,5)P₂ derivatives.

Scheme 2

Synthesis of PtdIns derivative PI-C15.