A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides

Abstract

Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly active THF from the freeze-tolerant beetle, Upis ceramboides, by means of ice affinity. Amino acid chromatographic analysis, polyacrylamide gel electrophoresis, UV-Vis spectrophotometry, and NMR spectroscopy indicated that the THF contained little or no protein, yet it produced 3.7 +/- 0.3 degrees C of TH at 5 mg/ml, comparable to that of the most active insect antifreeze proteins. Compositional and structural analyses indicated that this antifreeze contains a beta-mannopyranosyl-(1-->4) beta-xylopyranose backbone and a fatty acid component, although the lipid may not be covalently linked to the saccharide. Consistent with the proposed structure, treatment with endo-beta-(1-->4)xylanase ablated TH activity. This xylomannan is the first TH-producing antifreeze isolated from a freeze-tolerant animal and the first in a new class of highly active THFs that contain little or no protein.

Fig. 1.

SDS/PAGE (12%) of R1 and R2 fractions. (A) Silver-stained gel. Lane assignments: 1, ice-purified R1; 2, low-molecular-weight standards. (B) Silver stained gel. Lane assignments: 1, low-molecular-weight standards; 2, ice purified R2. (C) Gel stained with Sypro Ruby. Lane assignments: 1, low molecular weight standards; 2, blank (loading dye only); 3, ice purified R1; 4, ice purified R2. Ice-purified R1 and R2 were applied to two additional lanes (lanes 5 and 6, respectively), which were excised from the gel. Each lane was divided into four segments and the THF was eluted in distilled water overnight. After dialysis, the sample was concentrated and TH was measured. TH values (°C) are shown for lanes 5 and 6 for each gel fragment.

Fig. 2.

UV absorbance spectra of R1 and R2 fractions compared with that of BSA. Squares, BSA at 0.12 mg/ml; circles, 1:100 dilution of R2; triangles, 1:100 dilution of R1.

Fig. 3.

A comparison of 600 MHz ¹H NMR spectra of THFs isolated by ice affinity from three successive extraction buffers. (A) Buffer R1, soluble fraction. (B) Buffer R2, first membrane-associated fraction. (C) Buffer R3, second membrane-associated fraction. Decreasing signal to noise indicates lower THF concentrations.

Fig. 4.

Partial 800 MHz ¹H NMR and TOCSY spectra of R1 showing correlations among lipid signals. (A) ¹H NMR spectrum showing lipid signals that correspond to crosspeaks in (B). Numbers below the bracketed regions indicate relative signal areas. (B) Cross-peaks (connected by dashed lines) in the TOCSY spectrum indicate spin connectivities between CH₃ and different types of -CH₂-protons in the fatty acid constituent of the R1 THF.

Fig. 5.

MALDI-TOF mass spectrum of R1. Red and blue brackets indicate ions separated by either the mass of an aldohexose (180.06–18.01 [reducing end H₂O] = 162.05 Da) or aldopentose (150.05–18.01 [reducing end H₂O] = 132.04 Da), respectively.

Fig. 6.

Partial 2D ¹H NMR spectra of R1 at 800 MHz. (A) Partial HSQC spectrum showing ¹H-¹³C correlations for the saccharide ¹H signals shown in (B). Black contours correlate ¹³C and methylene (-CH₂-) protons, and red contours correlate ¹³C to methine (-CH =) or methyl (-CH₃) protons. Crosspeak assignments for the Manp and Xylp constituents are shown as M1-M6′ and X1-X5′, respectively. (B) Partial 1D ¹H NMR spectrum showing saccharide signals observed for R1. (C) Expansion of the anomeric signals observed in the HSQC spectrum. (D) Partial HSQC-TOCSY spectrum showing proton signals in (B) that correlate with the anomeric signals observed in (C), allowing identification of most of the ring protons in Xylp, but only H2 in Manp because of the small ³J_H1,H2 and ³J_H2,H3 values in Man residues.

Fig. 7.

Proposed disaccharide core structure comprising the THF isolated from U. ceramboides.