neo-Inositol Polyphosphates in the AmoebaEntamoeba histolytica

Abstract

We have reexamined the structure of inositol phosphates present in trophozoites of the parasitic amoeba Entamoeba histolytica and show here that, rather than being myo-inositol derivatives (Martin, J.-B., BakkerGrunwald, T., and Klein, G. (1993) Eur. J. Biochem. 214, 711-718), these compounds belong to a new class of inositol phosphates in which the cyclitol isomer is neoinositol. The structures of neo-inositol hexakisphosphate, 2-diphospho-neo-inositol pentakisphosphate, and 2,5-bisdiphospho-neo-inositol tetrakisphosphate, which are present in E. histolytica at concentrations of 0.08 -0.36 mM, were solved by two-dimensional 31 P-1 H NMR spectroscopy. No evidence for the co-existence of their myo-inositol counterparts has been found. These neoinositol compounds were not substrates of 6-diphosphoinositol pentakisphosphate 5-kinase, an enzyme purified from Dictyostelium discoideum that phosphorylates 6-diphospho-myo-inositol pentakisphosphate and more slowly also myo-inositol hexakisphosphate, specifically on position 5. Because preliminary data indicate that large amounts of the same neo-inositol phosphate and diphosphate esters are also present in another primitive amoeba, Phreatamoeba balamuthi, the occurrence of high concentrations of neo-inositol polyphosphates may be much more general than previously thought.Inositol phosphates are members of a large family of naturally occurring compounds that because of their complex role in cell signaling and homoeostasis have been intensively studied during the last two decades (1). Research has focused mainly on the commonly known myo-derivatives, although many other naturally occurring inositol stereoisomers are known. The most abundant and ubiquitous member of the family, myo-InsP 6 , has been shown to reach an intracellular concentration close to 1 mM in Dictyostelium discoideum (2, 3). Recently, new members of this class of compounds have been identified in cellular slime molds as 5-PP-and 6-PP-myo-InsP 5 1 and 3,5-bis-PP-and 5,6-bis-PP-myo-InsP 4 (4 -6). The same or similar highly phosphorylated and diphosphorylated compounds have been detected in free living and parasitic amoebae and a number of mammalian cell types (7-11). The latter molecules may function in cell signaling (2,(12)(13)(14). Entamoeba histolytica is a human intestinal parasite that causes amoebiasis. Its trophozoites contain high amounts of inositol polyphosphates (8). myo-Inositol is the major cyclitol building natural inositol polyphosphates, and other isomers are only rarely found and in very low concentrations. Consequently, the two major inositol phosphates of E. histolytica have been preliminarily identified as myo-inositol 2,4,6-triphosphate and 5-PP-myo-InsP 5 (8). However, we subsequently noticed during comparative studies by high resolution anion exchange chromatography that the inositol polyphosphates from E. histolytica were not eluted exactly with the same retention time as the myo-inositols used as reference. This casted some doubt on the previous identification. Therefore, we have r...

Inositol 1,3,4-Trisphosphate 5/6-Kinase Is a Protein Kinase That Phosphorylates the Transcription Factors c-Jun and ATF-2

“…Soluble extract from 293 cells induced for 3 days with tetracycline was fractionated on a heparin-agarose column followed by a Mono Q column. Fractions from the Mono Q column (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) were assayed for phosphorylation of Ins(1,3,4)P 3 and GST-AFT-2 (Fig. 5A).…”

“…The two tetrakisphosphate products are produced in equal amounts from Ins(1,3,4)P 3 , but when Ins(1,4,5)P 3 is the substrate only Ins(1,3,4,5)P 4 is a product. The activity of the amoebae enzyme is very low compared with that of the human and plant enzymes, which may be because of the fact that the inositol polyphosphates found in E. histolytica are not myo-derivatives but are neo-derivatives (14). In addition to producing two distinct isomers of InsP 4 from a single substrate, it has been shown recently that 5/6-kinase can utilize inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P 4 ) as a substrate for 1-kinase activity to produce InsP 5 (15).…”

Inositol phosphates in the environment

“…d-chiro-IP 6 was reported in pine needles (L'Annunziata & Fuller 1971), but this was subsequently questioned on analytical grounds (Cosgrove 1980). Recently, neo-inositol phosphates have been identified in trophozoites of the parasitic amoebae Entamoeba histolytica and Phreatamoeba balamuthi using two-dimensional nuclear magnetic resonance (NMR) (Martin et al 2000). However, the fact that (i) terrestrial plants only contain the myo isomer of IP 6 and (ii) chemical epimerization of myo-IP 6 has been ruled out, suggest that microorganisms must play a key role in the synthesis of IP 6 isomers other than myo-IP 6 in the soil (Cosgrove 1969b).…”

The “Other” Inositols and Their Phosphates: Synthesis, Biology, and Medicine (with Recent Advances inmyo‐Inositol Chemistry)

“…One has to point to these emerging myo-inositol pyrophosphates and their complexity as an area of great current chemical and biological activity and potential, but also note that neo-inositol IP 7 and IP 8 pyrophosphates are already known from protozoa, but are still of unknown function. [192] There are thus tantalising hints of wider activity already in evidence.…”

“…[192] The synthesis of neo-Ins(1,2,3,4,5,6)P 6 (137) and subsequent degradation to other neo-inositol phosphates by phytases has been described. [172] 9 muco-Inositol…”