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, 284 (25), 16822-31

Cryoprotectant Biosynthesis and the Selective Accumulation of Threitol in the Freeze-Tolerant Alaskan Beetle, Upis Ceramboides

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Cryoprotectant Biosynthesis and the Selective Accumulation of Threitol in the Freeze-Tolerant Alaskan Beetle, Upis Ceramboides

Kent R Walters Jr et al. J Biol Chem.

Abstract

Adult Upis ceramboides do not survive freezing in the summer but tolerate freezing to -60 degrees C in midwinter. The accumulation of two cryoprotective polyols, sorbitol and threitol, is integral to the extraordinary cold-hardiness of this beetle. U. ceramboides are the only animals known to accumulate high concentrations of threitol; however, the biosynthetic pathway has not been studied. A series of (13)C-labeled compounds was employed to investigate this biosynthetic pathway using (13)C{(1)H} NMR spectroscopy. In vivo metabolism of (13)C-labeled glucose isotopomers demonstrates that C-3-C-6 of glucose become C-1-C-4 of threitol. This labeling pattern is expected for 4-carbon saccharides arising from the pentose phosphate pathway. In vitro experiments show that threitol is synthesized from erythrose 4-phosphate, a C(4) intermediate in the PPP. Erythrose 4-phosphate is epimerized and/or isomerized to threose 4-phosphate, which is subsequently reduced by a NADPH-dependent polyol dehydrogenase and dephosphorylated by a sugar phosphatase to form threitol. Threitol 4-phosphate appears to be the preferred substrate of the sugar phosphatase(s), promoting threitol synthesis over that of erythritol. In contrast, the NADPH-dependent polyol dehydrogenase exhibits broad substrate specificity. Efficient erythritol catabolism under conditions that promote threitol synthesis, coupled with preferential threitol biosynthesis, appear to be responsible for the accumulation of high concentrations of threitol (250 mm) without concomitant accumulation of erythritol.

Figures

FIGURE 1.
FIGURE 1.
13C{1H} NMR spectrum of native hemolymph from winter-acclimatized U. ceramboides. A, partial spectrum (150 MHz) showing signal assignments for threitol (a), sorbitol (b), proline (c), and trehalose (d). B and C are expansions of A showing weak signals tentatively assigned to betaine-like compounds based on their distinctive chemical shifts and multiplicities.
FIGURE 2.
FIGURE 2.
Primary and secondary alcohol 13C signals in native hemolymph from winter-acclimatized U. ceramboides. A and B, expansions of the spectrum shown in Fig. 1. A, expansion showing secondary alcohol (-CHOH) carbons. B, expansion showing primary alcohol (-CH2OH) carbons. C and D correspond to A and B, respectively, after the sample was sequentially spiked with the following alditol standards: [1-13C]erythritol, [1-13C]glycerol, [1-13C]threitol, and natural abundance erythritol and ribitol. Spiking led to the following signal assignments: threitol (a), sorbitol (b), trehalose (c), erythritol (d), ribitol (e), glycerol (f), and ethylene glycol (g).
FIGURE 3.
FIGURE 3.
Metabolic fate in vivo of 13C arising from singly labeled glucose isotopomers. A, time course showing metabolism of 13C-enriched glucose isotopomers over 168 h at −4 °C: glucose (squares), sorbitol (triangles), and all remaining saccharides (circles). The relative contribution of each saccharide to the 13C pool was calculated by integrating all signals within a spectrum. Signal integrals from a given molecule were compared with determine how much a specific site within the molecule (e.g. C-1, C-2, etc.) was enriched above natural abundance. This was not possible for ethylene glycol because it exhibits only one signal; thus, the enrichment was estimated based on the ratio of enriched to natural abundance threitol signals. The enrichment of individual saccharides, usually in terms of a standardized signal area, was then summed across saccharides to determine the proportional contribution of each. B and C, the proportion of the total 13C enrichment represented by a given saccharide at 168 h after the injection of singly labeled glucose 13C-labeled isotopomers labeled at C-1—C-2 (B) and C-3—C-6 (C). Asterisks indicate saccharides that comprised significantly different (p < 0.05, Student's t test, n = 10) proportions of the total 13C pool with the injection of C-1—C-2 versus C-3—C-6 glucose 13C-labeled isotopomers.
FIGURE 4.
FIGURE 4.
Relative tetritol catabolism in vivo at low temperature. A, partial 13C{1H} NMR spectrum (150 MHz) of an ∼equimolar mixture of [1-13C]erythritol and [1-13C]threitol showing signals arising only from the labeled carbons. B and C, the mixture in A was injected into the hemolymph of a deacclimated U. ceramboides and held either for 6 days at 0 °C (B) or 5 days at 4 °C (C). The homogenates of two insects are pooled for each sample. Signal assignments are as follows: C-1 threitol (a), C-1 erythritol (b), and ethylene glycol (c).
FIGURE 5.
FIGURE 5.
In vivo tetritol catabolism at 23 °C. Equivalent amounts of [1-13C]erythritol (A) and [1-13C]threitol (B) were injected into the hemolymph of deacclimated U. ceramboides, which were held at 23 °C for 2, 8, 24, or 48 h before the homogenized insects were analyzed by 13C{1H} NMR. Signal intensities of labeled alditols observed in the homogenate were standardized against the C-1 signal of [1-13C]glycerol, which was added after heat inactivation of the homogenate. The standardized mean signal intensities ± S.D. of threitol (diamonds), erythritol (circles), and ethylene glycol (squares) are plotted against time. The sample size at each time point is from 1–4 individuals. (A) The 8 and 48 h time points represent only one individual. The initial time point (t = 0) for both alditols is estimated assuming 60% recovery of the labeled signal.
FIGURE 6.
FIGURE 6.
In vitro metabolism at −2.5 °C of putative intermediates in threitol biosynthesis. A and B, time course of signal intensities (relative to the internal standard) arising from in vitro metabolism of d-[1-13C]E4P (A) and d-[1-13C]T4P (B). The concentration of the internal standard was ∼2-fold higher in the T4P samples (relative to E4P samples), and both aldose phosphate stock solutions contained Eu4P arising from nonenzymatic isomerization prior to their addition to the homogenate. Only the total intensity of the erythritol and threitol signals was known at 24 h because the spectra were collected at −1 °C and their signals were coincident. Thus, the relative intensities at 24 h are estimates based on the total intensity. Subsequent spectra were collected at 22 °C, where the signals were well resolved. The number on each curve corresponds to a structure in the metabolic scheme (C). An asterisk indicates the site of 13C enrichment. Assignments are as follows: open diamonds, d-[1-13C]E4P (1); closed triangles, d-[1-13C]erythritol 4-phosphate (2); open circles, d-[1-13C]erythritol (3); closed squares, d-[1-13C]Eu4P (4); closed diamonds, d-[1-13C]T4P (5); asterisks, d-[1-13C]threitol 4-phosphate (6); closed circles, d-[1-13C]threitol (7).
SCHEME 1.
SCHEME 1.
Proposed alditol biosynthetic pathway in U. ceramboides. Metabolic scheme showing glycolysis and the pentose phosphate pathway. Biosynthetic pathway leading to threitol is in bold. Bold numbers indicate alternative metabolism pathways of E4P discussed under “Discussion.” Question marks indicate uncertainty associated with the mechanism of conversion of E4P to T4P. Eol4P, erythritol 4-phosphate; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; GAP, glyceraldehyde phosphate; Ru5P, ribulose 5-phosphate; Xu5P, xylulose 5-phosphate; Tol4P, threitol 4-phosphate. Ribulose 5-phosphate may first be isomerized to ribose 5-phosphate or reduced directly and dephosphorylated to form ribitol.

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