Harnessing 13C-labeled myo-inositol to interrogate inositol phosphate messengers by NMR

Abstract

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are an important group of metabolites and mediate a wide range of processes in eukaryotic cells. To elucidate the functions of these molecules, robust techniques for the characterization of inositol phosphate metabolism are required, both at the biochemical and the cellular level. Here, a new tool-set is reported, which employs uniformly ¹³C-labeled compounds ([¹³C₆]myo-inositol, [¹³C₆]InsP₅, [¹³C₆]InsP₆, and [¹³C₆]5PP-InsP₅), in combination with commonly accessible NMR technology. This approach permitted the detection and quantification of InsPs and PP-InsPs within complex mixtures and at physiological concentrations. Specifically, the enzymatic activity of IP6K1 could be monitored in vitro in real time. Metabolic labeling of mammalian cells with [¹³C₆]myo-inositol enabled the analysis of cellular pools of InsPs and PP-InsPs, and uncovered high concentrations of 5PP-InsP₅ in HCT116 cells, especially in response to genetic and pharmacological perturbation. The reported method greatly facilitates the analysis of this otherwise spectroscopically silent group of molecules, and holds great promise to comprehensively analyze inositol-based signaling molecules under normal and pathological conditions.

Fig. 1. Methods for the analysis of inositol polyphosphates. (a) Radioactively labeled InsPs can be resolved via SAX-HPLC and the amount of radioactivity in each fraction is analyzed. Alternatively, InsPs are enriched with TiO₂-beads from complex mixtures, subsequently resolved via PAGE, and detected by staining with toluidine blue. (b) ¹³C-labeled InsPs can be directly measured in complex samples using NMR spectroscopy.

Fig. 2. Enzymatic synthesis of [¹³C₆]myo-inositol followed by derivatization and purification. (i) MOPS pH 6.5, creatine phosphate, ATP, DTT, MgCl₂, hexokinase, creatine kinase in H₂O at rt. (ii) Tris pH 8.0, NAD⁺, NaCl, MgCl₂, inositol phosphate synthase in H₂O at 80 °C. (iii) Glycine pH 9.8, ZnCl₂, alkaline phosphatase in H₂O at 35 °C. (iv) Ac₂O in pyridine at 120 °C. (v) NaOMe in MeOH at rt.

Fig. 3. Synthesis and characterization of [¹³C₆]inositol polyphosphates. (a) Reagents and conditions: (i) CSA, trimethyl orthobenzoate, TEA in DMSO at 80 °C; 85% yield (ii) TFA in H₂O at rt; quantitative yield (iii) 5-phenyltetrazole, xylyl phosphoramidite, mCPBA in DCM at rt; 79% yield (iv) Pd(OH)₂/C in MeOH/H₂O at rt; 88% yield (v) NH₃ (aq.) at 60 °C; 65% yield (vi) xylyl phosphoramidite, 1H-tetrazole, mCPBA in DCM at rt; 38% yield (vii) Pd(OH)₂/C in MeOH/H₂O at rt; 83% yield (viii) MES pH 6.4, NaCl, ATP, creatine phosphate, MgCl₂, DTT, IP6KA in H₂O at 37 °C; quantitative yield (b) ¹H,¹³C-HMQC spectrum of [¹³C₆]InsP₅ at 5 μM. (c) ¹H,¹³C-HMQC spectrum of [¹³C₆]InsP₆ at 5 μM. (d) ¹H,¹³C-HMQC spectrum of [¹³C₆]5PP-InsP₅ at 5 μM. The positions of the carbon atoms and the solvent signal are indicated.

Fig. 4. Kinetic characterization and inhibition of IP6K1. (a) Michaelis–Menten kinetics for IP6K1. The initial velocity was measured in triplicate at different ATP concentrations and fitted against a model for substrate inhibition. (b) The inhibitory effects of TNP (N²-(m-trifluorobenzyl)-N⁶-(p-nitrobenzyl)purine), Myricetin, and Wortmannin were tested and the IC₅₀ values were determined. The ATP concentration was 2.5 mM for all inhibitor measurements (‡ enzyme activity at 0 μM inhibitor).

Fig. 5. Metabolic labeling of mammalian cell line HCT116, followed by NMR analysis. (a) General workflow for the preparation of whole cell extracts for NMR spectroscopy. (b) Left: ¹H,¹³C HMQC spectrum of an HCT116 extract. The peaks from 80 to 100 ppm in the F1 and between 0 and 3 ppm are folded into an empty region of the spectrum. Right: the inositol phosphate region of the spectrum is depicted in more detail. The identified InsPs are annotated while peaks that exhibit the expected splitting pattern in the carbon dimension but could not be attributed to either myo-inositol (Ins), InsP₅, InsP₆, nor 5PP-InsP₅ are highlighted by red arrows.

Fig. 6. Changes in 5PP-InsP₅ levels can be observed by NMR spectroscopy. (a) ¹H,¹³C HMQC spectrum of a HCT116 wt cell extract. (b) ¹H,¹³C HMQC spectrum of an extract of HCT116 wt cells treated with 10 mM NaF before extraction. (c) ¹H,¹³C HMQC spectrum of HCT116 PPIP5K^–/– cell extract. The inserts display triplicates of absolute cellular concentration of InsP₅, InsP₆ and 5PP-InsP₅ based on packed cell volume.