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Enhancement of Insect Antifreeze Protein Activity by Solutes of Low Molecular Mass

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Enhancement of Insect Antifreeze Protein Activity by Solutes of Low Molecular Mass The Journal of Experimental Biology 201, 2243–2251 (1998) 2243 Printed in Great Britain © The Company of Biologists Limited 1998 JEB1557 ENHANCEMENT OF INSECT ANTIFREEZE PROTEIN ACTIVITY BY SOLUTES OF LOW MOLECULAR MASS NING LI, CATHY A. ANDORFER AND JOHN G. DUMAN* Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA *Author for correspondence (e-mail: [email protected]) Accepted 20 May; published on WWW 14 July 1998 Summary Antifreeze proteins (AFPs) lower the non-equilibrium Glycerol is the only one of these enhancing solutes that is freezing point of water (in the presence of ice) below the known to be present at these concentrations in melting point, thereby producing a difference between the overwintering D. canadensis, and therefore the freezing and melting points that has been termed thermal physiological significance of most of these enhancers is hysteresis. In general, the magnitude of the thermal unknown. The mechanism(s) of this enhancement is also hysteresis depends upon the specific activity and unknown. concentration of the AFP. This study describes several low- The AFP used in this study (DAFP-4) is nearly identical molecular-mass solutes that enhance the thermal hysteresis to previously described D. canadensis AFPs. The mature activity of an AFP from overwintering larvae of the beetle protein consists of 71 amino acid residues arranged in six 12- Dendroides canadensis. The most active of these is citrate, or 13-mer repeats with a consensus sequence consisting of which increases the thermal hysteresis nearly sixfold from Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X13, where 1.2 °C in its absence to 6.8 °C. Solutes which increase X3 and X11 tend to be charged residues, X5 tends to be Thr activity approximately fourfold are succinate, malate, or Ser, X6 to be Asn or Asp, X9 to be Asn or Lys and X13 to aspartate, glutamate and ammonium sulfate. Glycerol, be Ala in the 13-mers. DAFP-4 is shorter by one repeat than sorbitol, alanine and ammonium bicarbonate increased previously described D. canadensis AFPs. thermal hysteresis approximately threefold. Interestingly, 0.5 mol l−1 sodium sulfate eliminated activity. Solute Key words: antifreeze protein, thermal hysteresis activity, antifreeze concentrations between 0.25 and 1 mol l−1 were generally protein activation, Dendroides canadensis, beetle, insect antifreeze required to elicit optimal thermal hysteresis activity. protein. Introduction Antifreeze proteins (AFPs), also known as thermal point, except for a small colligative effect. This produces a hysteresis proteins, were first identified in polar marine fishes difference between the freezing and melting points which has where they function to depress the freezing point of the body been termed thermal hysteresis (DeVries, 1971, 1986). While fluids of the hypo-osmoregulating fish below that of sea water the structures of the various AFPs differ considerably, their (DeVries, 1971; DeVries and Cheng, 1992; Davies and Hew, general mechanism of freezing point depression depends on the 1990). AFPs are also present in certain terrestrial arthropods, ability to adsorb onto the surface of potential seed ice crystals, including insects (Duman, 1977; Duman et al. 1993), spiders probably via hydrogen bonding. This forces crystal growth into (Duman, 1979a; Husby and Zachariassen, 1980), mites (Block highly curved (high free energy) fronts, rather than the and Duman, 1989) and centipedes (Tursman et al. 1994; preferred low-curvature (low free energy) fronts. Therefore, Tursman and Duman, 1995). More recently, thermal hysteresis growth is halted by the Kelvin effect until the temperature is proteins have been identified in many plants (Urrutia et al. lowered sufficiently (Raymond et al. 1977, 1989; Knight et al. 1992; Griffith et al. 1992a,b; Duman and Olsen, 1993; Duman, 1991; DeVries and Cheng, 1992). While details of the 1994), fungi and bacteria (Duman and Olsen, 1993). In adsorption mechanism are somewhat uncertain, considerable overwintering larvae of the beetle Dendroides canadensis, the amounts of data are now available on the fish AFPs (Sicheri AFPs function to inhibit inoculative freezing across the cuticle and Yang, 1995), and a lattice match between polar side- by external ice (Olsen et al. 1998) and to inhibit ice nucleators groups of the AFP and water molecules in the ice crystal both in the gut and in hemolymph (Olsen and Duman, appears likely. 1997a,b). Four basic categories of fish AFPs have been identified, AFPs depress the non-equilibrium freezing point of water by one glycoprotein type with considerable repeating structure a non-colligative mechanism while not lowering the melting and three protein (peptide) types with varying levels of 2244 N. LI, C. A. ANDORFER AND J. G. DUMAN repeating structure, presumably to allow efficient hydrogen bonding to the crystal lattice of ice (for reviews, see DeVries 0.4 and Cheng, 1992; Davies and Hew, 1990). Three of the AFPs 14.75 from the larvae of the beetle Dendroides canadensis have been characterized (Duman et al. 1998). These are approximately 8.7 kDa in molecular mass and consist of 11.07 seven 12- or 13-mer repeat units with a consensus sequence 3.20 of C-T-X3-S-X5-X6-C-X8-X9-A-X11-T-X13, where X3 and X11 0.2 22.52 tend to be charged residues, X5 tends to be threonine or serine, 12.80 X6 to be asparagine or aspartate, X9 to be asparagine or lysine 17.42 and X13 to be alanine in the 13-mers. Through most of the 2.45 length of the proteins, every sixth residue is a cysteine, and 23.76 all these residues are involved in disulfide bridges (Li et al. 7.54 1998). The AFPs from the larvae of the beetle Tenebrio Absorbance at 230 nm (arbitrary units) molitor are quite similar to those of D. canadensis, including 10 20 30 40 complete identity of the cysteine residues (Graham et al. 1997). Although the AFP from the spruce budworm Retention time (min) Choristoneura fumiferana has 12-mer repeating units, this Fig. 1. Reverse-phase HPLC of the peptide mixture resulting from AFP is not similar to the D. canadensis AFPs (Tyshenko et trypsin digestion of DAFP-4. Conditions used were as stated in the al. 1997). text. DAFP-4 elutes at 22.46 min. In general, the magnitude of the thermal hysteresis activity of AFPs depends upon the specific activity of the particular AFP and the AFP concentration, up to a point where the 4) used in this study elutes from the reverse-phase high- activity plateaus. While the blood of AFP-producing fish may performance chromatography (RP-HPLC) column used in the exhibit up to approximately 2 °C of thermal hysteresis final step of the purification at 22.46 min. activity, a value sufficient to prevent freezing in ice-laden sea water, it is common for the hemolymph of insects to have Peptide sequencing of DAFP-4 considerably greater activity. Overwintering larvae of D. DAFP-4 (200 µg) was digested with 8 µg of trypsin canadensis routinely have a mean thermal hysteresis activity (13 000 international units mg−1; Sigma Chemical Co.) in 80 µl of 5–6 °C in the hemolymph, and some individuals may have of 50 mmol l−1 Tris–HCl, pH 7.0 at 37 °C, for 24 h. The an activity of 8–9 °C (Duman, 1980). Therefore, it was digestion mixture of peptides was separated using a Rainin perplexing when purified D. canadensis AFP, even at very reverse-phase C18 HPLC column (4.6 mm×100 mm) with a high protein concentrations, produced much less activity than 0.1 % trifluoroacetic acid/acetonitrile gradient of 5 % to 30 % that seen in the hemolymph (Wu et al. 1991a). However, an in 40 min (Fig. 1). Peptides 3.20, 11.07, 14.75 and 22.52 were endogenous activator (enhancer) protein was later purified each fully reduced in 0.1 mol l−1 sodium citrate, pH 3.0, with (Wu and Duman, 1991). Addition of the activator protein to 15 mmol l−1 Tris(2-carboxyethyl)phosphine hydrochloride a solution containing 4 mg ml−1 of D. canadensis AFP raised (TCEP) at 60 °C for 20 min. The reduced peptides were the thermal hysteresis activity from 1.60 °C to 5.24 °C. This separated again by RP-HPLC and sequenced on a Beckman LF activator appears to function by binding to the AFP, or vice 3000 protein sequencer. versa, such that the AFP–activator complex can still hydrogen Peptide 14.75 had a blocked N terminus which was bond to ice but now blocks a larger surface area of the deblocked by treatment with pyroglutamyl peptidase potential seed ice crystal and/or is more difficult to overgrow, (Tsunasawa and Hirano, 1993) and then sequenced. so that the hysteretic freezing point is further depressed (Wu and Duman, 1991; Wu et al. 1991b). Studies reported here Mass spectrometry analysis identify certain low-molecular-mass solutes which Matrix-assisted laser desorption ionization time-of-flight demonstrate unusual abilities to increase the thermal (MALDI-TOF) mass spectra were obtained on a PerSeptive hysteresis activity of a D. canadesis AFP. Since the AFP used Biosystems/Voyager-DE MALDI-TOF mass spectrometer. in this study has not been described previously, information Matrix (α-cyano-4-hydroxycinnamic acid) was prepared as a on the sequence and stability of this D. canadensis AFP saturated solution in 1:1 (v/v) water:acetonitrile with 0.1 % (DAFP- 4) is also presented. trifluoroacetic acid (TFA). The sample was dissolved in double-distilled H2O–TFA at pH 3.0. A 0.5 µl portion of sample solution was then mixed with an equal volume of the Materials and methods saturated matrix solution in a sample plate and allowed to air- Purification of Dendroides canadensis AFP-4 dry before analysis.
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