The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones

Abstract

Inositol phosphates (InsPs) are ubiquitous in all eukaryotes. However, since there are 63 possible different phosphate ester isomers, the analysis of InsPs is challenging. In particular, InsP₁, InsP_2, and InsP₃ already amass 41 different isomers, of which some occur as enantiomers. Profiling of these "lower" inositol phosphates in mammalian tissues requires powerful analytical methods and reference compounds. Here, we report an analysis of InsP₂ and InsP₃ with capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS). Using this method, the bacterial effector RipBL1 was analyzed and found to degrade InsP₆ to Ins(1,2,3)P₃, an understudied InsP₃ isomer. This new reference molecule then aided us in the assignment of the isomeric identity of an InsP₃ while profiling human samples: in urine and kidney stones, we describe for the first time the presence of defined and abundant InsP₃ isomers, namely Ins(1,2,3)P₃, Ins(1,2,6)P₃ and/or Ins(2,3,4)P₃.

Fig. 1. Separation of InsPs by CE-ESI-MS. (A) Structures of commercial InsP₂ and InsP₃ isomers. Achiral isomers are labeled as “meso”. (B and C) Separation of InsP₂ and InsP₃ standards by CE-ESI-MS, BGE: 35 mM ammonium acetate titrated with ammonium hydroxide to pH 9.75. (D) Separation of InsP₂ and InsP₃ standards by CE-ESI-MS, BGE: 50 mM ethylamine titrated with formic acid to pH 10.0.

Fig. 2. Identification of the InsP₃ (black line) dephosphorylation product of [¹³C₆] InsP₆ by the RipBL1 enzyme. (A) CE-ESI-MS analysis of InsP₃ individually spiked (red line) with Ins(3,4,5)P₃ standard, Ins(1,4,6)P₃ standard, Ins(1,3,4)P₃ standard, Ins(2,4,5)P₃ standard, Ins(1,4,5)P₃ standard, Ins(1,2,6)P₃ standard, Ins(1,3,5)P₃ standard or Ins(2,3,5)P₃ standard, as indicated. (B) Analysis of InsP₂ generated from Ins(3,4,5)P₃ and the [¹³C₆] InsP₃ isomer by heating to 100 °C for 2.5 h. Extracted ion electropherograms of [¹³C₆]-labelled InsP₂ (black lines) generated by [¹³C₆] InsP₃ and InsP₂ (red trace) generated by Ins(3,4,5)P₃. (C) [¹³C₆] InsP₂ (black line) generated from [¹³C₆] InsP₃ after heating to 100 °C for 2.5 h spiked with Ins(1,2)P₂ standard or Ins(1,3)P₂ standard as indicated (red line). Note that the [¹³C₆] InsP₂-1 isomer has the same migration time as the Ins(1,2)P₂ standard and that the [¹³C₆] InsP₂-2 isomer has the same migration time as the Ins(1,3)P₂ standard.

Fig. 3. Profiling of InsP_s in human kidney stones. (A) Extraction and analysis workflow of kidney stones for CE-ESI-MS. (B) InsPs distribution in kidney stone 1 (contains 80% COM and 20% COD), kidney stone 2 (contains 70% CaHPO₄ and 30% COM) and kidney stone 3 (contains 20% COM and 80% COD) from three different patients. (C) Extracted ion electropherograms of [¹³C₆] Ins(1,2,3)P₃ (black line) and InsP₃ in kidney stone 1 (red area). (D) Extracted ion electropherograms of [¹³C₆] Ins(1,2,3)P₃ (black line) and InsP₃ in kidney stone 2 (red area).

Fig. 4. InsPs in urine samples from patients with kidney stones vs. healthy people. (A) InsPs distribution in urine samples: ten samples from healthy individuals, and nine samples from patients with kidney stones. (B) Extracted ion electropherograms of [¹³C₆] Ins(1,2,3)P₃ (black line) and InsP₃ in urine from patients (red trace). (C) Extracted ion electropherograms of [¹³C₆] Ins(1,2,3)P₃ (black line) and InsP₃ in urine from healthy individuals (red trace).