Tài liệu Báo cáo khoa học: Hyperactive antifreeze protein in flounder species The sole freeze protectant in American plaice docx

Hyperactive antifreeze protein in flounder species The sole freeze protectant in American plaice Sherry Y. Gauthier 1 , Christopher B. Marshall 1 , Garth L. Fletcher 3 and Peter L. Davies 1,2 1 Department of Biochemistry, Queen’s University, Kingston, ON, Canada 2 Protein Function Discovery Group, Queen’s University, Kingston, ON, Canada 3 Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, NF, Canada Antifreeze proteins (AFPs) are functionally defined by their ability to bind to the surface of ice and inhibit its growth, which causes a lowering of the freezing tem- perature below the equilibrium freezing ⁄ melting point [1]. This thermal hysteresis (TH) effect of AFPs enables teleost fishes to live in ice-laden polar and subpolar oceans where temperatures can reach the freezing point of the seawater ()1.9 °C), which is over 1 C° colder than the freezing temperature of their hypotonic body fluids ()0.7 to )0.9 °C). Without the protection of AFPs, fish are effectively supercooled in these waters and will freeze on contact with ice [2,3]. Thus, the recent acquisition of AFPs during the late stages of the teleost radiation has enabled some species to survive in and ⁄ or expand into the relatively new niche created by sea-level glaciation 1–20 million years ago [4]. Type I AFPs are small, monomeric, alanine-rich single a-helices that have an 11-amino-acid periodicity. They are one of five distinct nonhomologous types of AFP found in fishes [5] and they are present in some righteye flounders, including the winter flounder (Pseudopleuronectes americanus), yellowtail flounder (Limanda ferruginea) and Alaskan plaice (Pleuronectes quadritaberulatus) [6–8]. The AFP isoforms in the win- ter flounder have been particularly well characterized. One of them, the 37-amino-acid HPLC-6, was the first AFP to have its structure solved [9,10] and to have the ice plane to which it binds defined by ice-etching [8]. The differences between isoforms mainly lie in their length (the number of 11-amino-acid repeats being either three or four) and in the amino acid replace- ments on the less well conserved hydrophilic side of Keywords alpha-helix; antifreeze protein; freezing point depression; ice Correspondence P. L. Davies, Department of Biochemistry, and Protein Function Discovery Group, Queen’s University, Kingston, ON, K7L 3N6, Canada Fax: +1 613 5332497 Tel: +1 613 5332983 E-mail: daviesp@post.queensu.ca (Received 24 March 2005, revised 8 July 2005, accepted 12 July 2005) doi:10.1111/j.1742-4658.2005.04859.x The recent discovery of a large hyperactive antifreeze protein in the blood plasma of winter flounder has helped explain why this fish does not freeze in icy seawater. The previously known, smaller and much less active type I antifreeze proteins cannot by themselves protect the flounder down to the freezing point of seawater. The relationship between the large and small antifreezes has yet to be established, but they do share alanine-richness (> 60%) and extensive a-helicity. Here we have examined two other right- eye flounder species for the presence of the hyperactive antifreeze, which may have escaped prior detection because of its lability. Such a protein is indeed present in the yellowtail flounder judging by its size, amino acid composition and N-terminal sequence, along with the previously character- ized type I antifreeze proteins. An ortholog is also present in American plaice based on the above criteria and its high specific antifreeze activity. This protein was purified and shown to be almost fully a-helical, highly asymmetrical, and susceptible to denaturation at room temperature. It is the only detectable antifreeze protein in the blood plasma of the American plaice. Because this species appears to lack the smaller type I antifreeze proteins, the latter may have evolved by descent from the larger antifreeze. Abbreviations AFP, antifreeze protein; ApAFP, American plaice antifreeze protein; IAP, ice affinity purification; TH, thermal hysteresis. FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS 4439 these amphipathic helices. Indeed, the conservation of the opposite alanine-rich side was helpful in identifying this as the ice-binding face of the AFP [11]. Two puzzling issues have persisted in this field for many years. One is that AFPs have not been found in some righteye flounders that inhabit the same geogra- phical range as AFP-producing species [12]. Here, an answer could be that fish living at greater depths are less likely to encounter ice and might be able to sur- vive in a supercooled state by avoiding nucleation. The other conundrum is that levels of type I AFP in the winter flounder ( 10 mgÆ mL )1 ) appear to be too low to guarantee protection down to the freezing point of seawater )1.9 °C [7]. The TH activity produced by this concentration of type I AFP is only 0.7 C°, which when added to the colligative freezing point depression of solutes in the blood (0.7–0.9 C°) fails to reach the critical )1.9 °C. We have solved the latter issue with the recent discovery in winter flounder blood plasma of a much larger dimeric (2 · 17 kDa ¼ 34 kDa) ala- nine-rich AFP that is extremely effective in TH and contributes more than enough activity to protect the fish to the freezing point of seawater [13,14]. The main reason this isoform has escaped detection for 30 years is that it is very thermolabile and denatures at room temperature. This discovery has prompted us to exam- ine the distribution of this new hyperactive AFP, which might have been missed in other species. The yellowtail flounder is known to produce type I AFP [7] but the presence of the larger, hyperactive AFP was not previously observed or anticipated. The American plaice (Hippoglossoides platessoides) has pre- viously been reported to exhibit antifreeze activity [12], but the AFP type that is present there was not identi- fied. In light of the recent discovery of the hyperactive but labile ‘5a-like’ 17 kDa AFP in winter flounder we have re-examined the American plaice. We note the absence of low M r AFPs, but have found a high M r form (ApAFP) with properties very similar to those of the newly discovered large flounder AFP. This ortho- log is very active and sufficiently abundant to protect the fish down to the freezing point of seawater. The absence of the small type I AFPs in the American plaice argues that these single helix antifreezes of the other flounder species may have their evolutionary ori- gins in the larger AFP. Results Purification of AFP from American plaice plasma Purification of American plaice antifreeze protein (ApAFP) from thawed blood plasma was initially done entirely by sequential size-exclusion chromatography. The first Sephadex G-75 column chromatography run at 4 °C produced a fairly typical A 230 profile for fish plasma, with most of the proteins eluting in the void peak (fractions 30–40 in Fig. 1A), but without any prominent smaller peaks in the region where the type I AFPs elute in winter flounder plasma [13]. A single peak of thermal hysteresis activity, which was associ- ated with spindle-shaped ice crystal morphology (Fig. 2A, upper inset), was found on the trailing edge of the void peak (fractions 40–45). There was no trace of activity thereafter, even in the region (fractions 70– 80) where type I AFPs would typically elute. The unfractionated plaice plasma had thermal hysteresis values of 700–1000 mOsm, which corresponds to 1.3– 1.8 C° (Fig. 2A). Allowing for dilution during the chromatography, the peak seen in Fig. 1A can account for the activity loaded onto the column. Rechromatography of the activity peak from Fig. 1A on the same Sephadex G-75 column at 4 °C gave an A 230 profile with two peaks, a void peak and a well-defined peak on the trailing edge (fractions 40–50) in the region where the thermal hysteresis activity was detected in the first column (Fig. 1B). Again, the ther- mal hysteresis activity was concentrated in just a few fractions (41–43). In a subsequent calibration chroma- tography of this column, horse myoglobin (M r 17 000) eluted around fraction 60 as shown by the vertical arrow. A third size-exclusion chromatography was done at room temperature on an FPLC apparatus using a Superose-12 column. Fractions 41–43 from Fig. 1B gave rise to one major peak around 12–12.5 mL with a prominent shoulder at around 11.5 mL (Fig. 1C). The thermal hysteresis activity was entirely concentrated under the shoulder on the leading edge of the peak (shown by the horizontal bar). Despite the short time- span of the chromatography, much of the ApAFP appears to have denatured to give rise to the large peak with a lower apparent M r . Calibration of the Superose-12 column with protein standards (Fig. 1C) showed that the TH-active shoulder chromatographed as if it were a 65–70 kDa globular protein (coeluted with BSA) and the inactive main peak behaved as a 37 kDa protein. However, MALDI mass spectrometry showed that only one species was present in both the peak and the shoulder. The mass of this protein was 17 843 Da (Fig. 3). Refinement of the purification procedure With the development of ice affinity purification [15] we were able to incorporate a different sequence of Hyperactive antifreeze protein in fish S. Y. Gauthier et al. 4440 FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS chromatography steps to avoid procedures that resul- ted in denaturation of the AFP. The initial size-exclu- sion chromatography on Sephadex G-75, followed by ice affinity purification and passage through DEAE- Sephacel, produced ApAFP that was extremely active and almost pure as judged by SDS ⁄ PAGE (Fig. 3, lane 3). ApAFP did not bind to the anion exchanger, pre- sumably because its pI is higher than the pH of the bicarbonate buffer (7.0). A B C Fig. 1. Purification of American plaice AFP by size-exclusion chro- matography. (A) Fractionation of American plaice plasma on a Sephadex G-75 column. (B) Refractionation of pooled active AFP fractions from (A) on Sephadex G-75. Absorbance at 230 nm (black dot) and thermal hysteresis activity (grey triangle) are shown across each of the elution profiles. Fraction volume (5 mL). (C) Chromato- graphy of pooled active AFP from (B) on an FPLC Superose-12 col- umn (absorbance at 280 nm vs. elution volume in mL). Active ApAFP fractions are indicated by the bar. Inset: calibration of the Superose-12 column (Log M r vs. elution volume) with protein standards (I: bovine gamma globulin, 158 kDa; II: bovine serum albumin, 67 kDa; III: horse skeletal muscle myoglobin, 17 kDa; IV: bovine heart cytochrome c, 12.3 kDa and V: type III AFP, 7 kDa [23]. The elution position of the main ApAFP peak is shown on the calibration curve by j and the higher molecular mass shoulder by h. B A Fig. 2. Thermal hysteresis activity of American plaice plasma and AFP as a function of dilution. (A) Serial twofold dilutions of plasma were made into 100 m M ammonium bicarbonate (pH 7.9). Data points are the average of two readings. Inset above the activity curve: image of the ice crystal shape obtained with hyperactive American plaice antifreeze protein. Inset below the activity curve: images of the crystal shapes obtained with hyperactive antifreeze proteins of winter flounder and yellowtail flounder compared to the bipyramidal shape produced by type I AFP. (B) Serial dilutions of pure ApAFP were made into 100 m M ammonium bicarbonate (pH 7.9) containing 1 mgÆmL )1 BSA. Data points are the average of at least two readings. Inset is an expansion of the data points for the low concentration readings. S. Y. Gauthier et al. Hyperactive antifreeze protein in fish FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS 4441 Characterization of ApAFP ApAFP migrated as a 17 kDa band on SDS ⁄ PAGE. This band was blotted onto a poly(vinylidene difluo- ride) membrane, excised, and sequenced by N-terminal Edman degradation (Table 1). The 12 residues determined were clearly homologous to, but distinct from, the start of the winter flounder 5a-like 17 kDa AFP and the theoretical gene product (5a) after which it was named [16]. For example, the seventh residue in ApAFP is Lys. The equivalent residue in 5a is also Lys but is Arg in the winter flounder 5a-like protein. The fifth, sixth and tenth residues in ApAFP are Gly, Thr and Ser, respectively, but are Ala in the alignment of the other two sequences. The mass of ApAFP determined by MALDI-TOF mass spectrometry was 17 843.33 ± 0.06 Da, which is 1160 Da larger than the 16 683 Da winter flounder 5a-like AFP (Fig. 3). Its amino acid composition is quite similar to that of the latter winter flounder AFP and to that of the predicted 16 267 Da product of the 5a gene (Table 2). In particular, all three proteins have Ala as the most abundant amino acid by a large mar- gin. It makes up 60–65% of the compositions of 5a and 5a-like protein and 55% of ApAFP. Of the other amino acids, Thr (13%) is the next most abundant, and there are very low amounts of aromatic and long- chain aliphatic amino acids. American plaice AFP is unusually active compared to other fish AFPs Amino acid analysis of ApAFP also provided an opportunity to correlate its TH activity with an accu- rately determined protein concentration. At a concen- tration of 0.4 mgÆmL )1 , ApAFP had a TH activity of 2.2 C°. This compares favourably with the winter Fig. 3. Comparison of hyperactive AFP orthologs in three righteye flounders. Mass spectra of yellowtail flounder (1), winter flounder (2) and American plaice (3) hyperactive AFPs shown above the SDS ⁄ PAGE analysis of the three samples. Approximately 1 lgof each protein was loaded on a 10% polyacrylamide SDS gel in the Tris ⁄ Tricine buffer system. M refers to the molecular mass markers of 26.6 kDa and 20 kDa. Table 1. N-terminal sequences of hyperactive AFPs from righteye flounders. Residue X at the N terminus of the 16 683 Da, winter flounder AFP could not be conclusively identified by Edman degra- dation but is likely to be Asn, Thr or Ala. The N-terminal sequence of the 5a hypothetical gene product was predicted from the previ- ously sequenced flounder gene, 5a [16], using a signal peptide pre- diction program. Sequence Source XIDPAARAAAAA Winter flounder AFP IDPAAKAAAAA 5a hypothetical gene product SIDPGTKAASAA American plaice AFP NIDPAVKAAAA Yellowtail flounder AFP Table 2. Amino acid compositions of the large, hyperactive AFPs from righteye flounders and the putative 5a gene product. Amino acid 5a gene product (mol%) Winter flounder (mol%) American plaice (mol%) (1st analysis) American plaice (mol%) (2nd analysis) Yellowtail flounder mol% Ala 64.6 60.6 55.8 59.6 61.5 Thr 13.3 9.3 13.9 13.9 10.7 Val 4.6 4.2 1.9 1.9 3.6 Asx 4.1 6.6 3.6 3.2 4.6 Ile 3.6 4.1 3.8 4.3 4.4 Lys 3.6 3.0 4.0 3.6 2.8 Glx 1.5 1.7 2.4 1.2 2.0 Ser 1.5 4.4 3.3 2.7 2.9 Pro 1.5 1.8 3.4 3.2 1.4 Leu 0.5 1.3 2.8 2.6 2.2 Phe 0 0.7 0.7 0.6 0.4 Tyr 0 0.6 0.4 0.2 0.4 Gly 1.0 0.5 2.6 1.8 2.5 Met 0 0.5 0.5 0.6 0.1 Arg 0 0.8 0.8 0.8 0.4 Total 99.8 100.1 99.9 100.2 99.9 Hyperactive antifreeze protein in fish S. Y. Gauthier et al. 4442 FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS flounder 5a-like AFP, which has 2.2 C° of TH activity at a protein concentration of 0.4 mgÆmL )1 [13]. The much weaker type I AFP (the 37 amino acid HPLC-6 isoform) [17] shows only  0.1 C° of TH at equivalent concentrations. The thermal hysteresis activity of American plaice plasma as a function of concentration shows the usual rectangular hyperbolic shape at high concentrations but the TH activity is unusually weak at low concentrations (Fig. 2A). This property has been observed for the 5a-like 17 kDa flounder AFP [14], although the explanation is not apparent. It could be that the AFP undergoes denaturation at low con- centrations, or it might act cooperatively and require a threshold concentration to begin to be effective at ice binding. To eliminate the possibility that denaturation occurs at low total protein concentrations, thermal hysteresis readings were done on dilutions of pure ApAFP in 1 mgÆmL )1 BSA. The plot of TH as a func- tion of ApAFP concentration with albumin present (Fig. 2B) has the same shape as the plasma dilution series (Fig. 2A), and the inset (Fig. 2B) shows the slow build up of activity at low AFP concentration that is peculiar to this antifreeze. Another diagnostic tool for classifying and compar- ing AFPs is the ice crystal morphology they produce during thermal hysteresis measurements. The ice crys- tals formed by ApAFP are spindle- or lemon-shaped (Fig. 2A, upper inset). They are thick in the centre but show a convex tapering off towards the crystal tips that lie on the c-axis. These crystals lack the well- defined facets seen with the much smaller type I AFPs, which produce the classic hexagonal bipyramid with flat surfaces and a 3.3 : 1 ratio of c-toa-axis lengths (Fig. 2A, lower inset). CD analysis The CD spectrum of a 3 lm sample collected at 4–5 °C between 260 and 180 nm resembled that of other alanine-rich a-helical proteins [14,18–20], with a maxi- mum near 190 nm followed by a negative minimum near 208 nm and a second more intense minimum at longer wavelength (Fig. 4A). The maximum ellipticity at 189.2 nm had an amplitude of 94 000 degÆcm )2 Ædmol )1 while the two minima occurred at 208.5 and 220.1 nm with amplitudes of 35 100 and 44 600 degÆcm )2 Ædmol )1 , respectively. The ratio of the two minima ([h] 220.1 ⁄ [h] 208.5 ) was 1.27, typical of alanine-rich helices (including other type I AFPs) at low temperature. Deconvolution by circular dichroism neural networks (CDNN) of the ApAFP CD spectra at 4–5 °C shows that the protein is almost fully a-helical (94.7% a-helix, 7% b-turn, 1.4% random coil, with no substantial con- tribution from b-sheet, 0.5%: total ¼ 103.6%). As the sample was warmed from 4 to 8 °C, there was a small decrease in the intensity of the spectrum; however, from 8to16°C there was a very large decrease in the ampli- tude of the signal (Fig. 4A). The magnitude of the maxi- mum at 189 nm decreased by 47% at 12 °C and 63% at 16 °C while the 221.5 nm minimum decreased by 42% B A Fig. 4. Circular dichroism spectra of American plaice AFP at low temperature and the effect of heating. (A) The averaged spectrum at 4–5 °Cofa3l M sample of ApAFP is shown in black. The sam- ple was then very slowly heated and averaged spectra are presen- ted for the following temperature ranges: 4–8 °C, red; 8–12 °C, green; 12–16 °C, yellow; 16–20 °C, blue; 20–24 °C, pink; 24–30 °C, cyan; 30–40 °C, grey; 40–50 °C, dark red; 50–60 °C, dark yellow; and 60–75 °C, dark blue. (B) In a separate experiment, the mean residue ellipticity (222 nm) was plotted against temperature as a sample was warmed (d) then subsequently cooled (n)asdes- cribed in Experimental procedures. The mean residue ellipticity following 40 h at 4 °C is indicated by a triangle. Also shown on this plot are the mean residue ellipticity (222 nm) values of winter flounder AFP as a function of temperature as a sample was warmed (ÆÆÆjÆÆÆ) then subsequently cooled (h) as described in Experimental procedures. The mean residue ellipticity following overnight incubation at 4 °C is indicated by a square. S. Y. Gauthier et al. Hyperactive antifreeze protein in fish FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS 4443 and 56% at these two temperatures, respectively. The ([h] 222 ⁄ [h] 208 ) ratio decreased as the ApAFP sample was warmed. At 16 °C, the protein had lost roughly half of its helicity (48.6% a-helix, 16.1% b-turn, 12.8% random coil, 9% b-sheet: total ¼ 86.4%). Changes in the magni- tude of the spectrum were small between 16 and 40 °C. [At room temperature (20–24 °C) helicity was 40.5% where the total for all structures was 88%.] However, the measured ellipticities underwent a second, rapid decrease in magnitude as the temperature increased above 40 °C. Between 4 and 40 °C, the spectra intersect near an isodichroic point at  201 nm with a magnitude of  )16 000 degÆcm )2 Ædmol )1 . Above 40 °C, the ellip- ticity at this wavelength decreased, possibly due to pro- tein precipitation. To better determine the temperature at which the protein melts, to investigate the nature of the trans- ition and to determine the extent to which denatura- tion is reversible, melting and renaturation curves of ApAFP were derived using CD spectroscopy (Fig. 4B). The ellipticity at 222 nm ([h] 222 ) was measured as the sample was warmed from 4 to 30 °C. The magnitude decreased slightly when the sample was warmed above 4 °C, although the first substantial changes occurred at 7 °C as the magnitude of the [h] 222 decreased by  5%. As the sample was warmed to 12 °C, there was a very large transition during which the [h] 222 decreased by 50%. The apparent T m of this transition was 9 °C. There was no appreciable further change in the signal as the sample was warmed from 13 to 30 °C. To investigate whether the denaturation of this extremely thermolabile protein is reversible, CD was measured while the sample was cooled back down to 4 °C over 2 h. At each temperature,  80% of the ellipticity that was lost upon warming was recovered, thus producing a renaturation curve that was remark- ably parallel to the melting curve. Because the [h] 222 was not completely restored at 4 °C on this short time- scale, the sample was allowed to renature at 4 °C for 40 h and the [h] 222 was measured again. At this point the [h] 222 was 96% of that initially produced by the sample at 4 °C. We have contrasted this denaturation ⁄ renaturation profile with that obtained using winter flounder 5a-like protein. The winter flounder AFP melts sharply at a higher temperature ( 20 °C) but does not renature as rapidly or completely as the ApAFP (dotted lines in Fig. 4B). Consistent with these differences in renatura- tion, ApAFP recovers much more activity (60–100% after 70 min at room temperature) than the winter flounder AFP does, but its TH values show consider- able deviations from reading to reading and sample to sample. Isolation of the hyperactive AFP from yellowtail flounder plasma A plasma sample collected from yellowtail flounder in March, when the seasonal production of AFP is still high, had 500 mOsm (0.93 C°) of TH activity. This TH value is lower than those of the winter flounder and American plaice plasmas, but higher than would be produced by type I AFP alone, particularly as yel- lowtail flounder type I AFPs are less active than those produced by the winter flounder [21]. The morphology of the ice crystals formed in the plasma was spindle- like and resembled that produced by the American plaice and winter flounder 17 kDa AFPs, albeit slightly more elongated (Fig. 2A). This elongation probably reflects the presence of type I AFP in the plasma sam- ple. The major protein present after purification migra- ted on SDS ⁄ PAGE marginally faster than the winter flounder AFP with an apparent M r of  16 kDa (Fig. 3, lane 1). The monomeric mass of this protein was 16 283.8 Da as determined by MALDI-TOF MS (Fig. 3). Like the 17 kDa winter flounder and Ameri- can plaice AFPs, the amino acid composition of this protein was rich in alanine (61.5%) and threonine (10.7%), and had few hydrophobic residues (Table 2). The N-terminal sequence (NIDPAVKAAAA) was also similar to those of the other two AFPs, but with a valine substitution preceding the basic residue (Table 1). Discussion Biochemical characterization of the American plaice AFP clearly shows that it is an ortholog of the newly discovered hyperactive (5a-like) 17 kDa AFP in winter flounder [13]. It has a similar monomeric mass (17 843 Da compared to 16 683 Da for the flounder protein) and, like the flounder protein, it has a similar, much larger apparent molecular mass (67 kDa) by size-exclusion chromatography, suggesting an extended conformation. Also, its amino acid composition, N-ter- minal sequence and the CD spectrum it produces are all characteristic of the new 5a-like flounder AFP. Another diagnostic feature is its thermolability, which was clearly demonstrated by the CD spectra and FPLC chromatography on Superose-12. Indeed, ApAFP denatures at 9 °C, making it even more labile than the homolog in winter flounder, which melts at 20 °C. Our interpretation of the FPLC profile is that the small residual amount of undenatured ApAFP gives rise to the high molecular mass shoulder with thermal hysteresis activity. Based on observations of the winter flounder protein this species is likely to be a Hyperactive antifreeze protein in fish S. Y. Gauthier et al. 4444 FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS rod-like dimer [14]. The main peak is probably dena- tured monomer, which explains its lower apparent molecular mass, its loss of activity, and the fact that both the main peak and the shoulder only contain one protein type, the 17 843 Da species. The room temperature lability of these hyperactive proteins can perhaps explain why they have been missed before now. Because the winter and yellowtail flounders also have the more resilient smaller type I AFPs, it is possible that any loss of activity of the larger AFP went unnoticed or was attributed to poor recovery of activity during purification. The thermal lability of the larger AFP contrasts with the ‘elasticity’ of the smaller AFP. Although these short, monomeric helical AFP are 50% denatured at room temperature they completely regain their helicity and activity on cooling [22]. Obviously, antifreeze activity can only be measured near 0 °C because ice has to be present for the assay. Purified ApAFP is also capable of regaining helicity upon cooling but this does not correlate with complete recovery of activity. This is probably due to inefficient reassembly of dimeric quaternary structure, a feature that appears to be required for TH activity in the winter flounder homolog. Nevertheless, the ther- mal denaturation of ApAFP, even though it occurs at a lower T m , is a more reversible process than that of the 17 kDa flounder AFP, as measured by both CD and TH activity. Until now, the American plaice was thought to have no, or low levels of, antifreeze activity in their blood plasma [12]. Indeed, because we are dealing with a wild species residing in different geographical locations, inhabiting a range of depths, it is entirely possible that some populations of American plaice produce little, if any, AFP. Extreme variation in AFP production has, for example, been seen before in ocean pout from two different locations [23]. However, because of the labil- ity of ApAFP, it is also likely that some loss of activity occurred on previous occasions during collection or assay of the plasma. Although the new AFP is 10–100-times more active in TH than the small type I AFPs, they are quite likely related as paralogs. The original 5a ‘pseudogene’ sequence, which is clearly homologous (based on its predicted N-terminal sequence) to the large hyperactive AFPs of winter flounder, American plaice and yellow- tail flounder was isolated from a winter flounder genomic library by hybridization to a type I AFP cDNA probe [16,24]. Moreover, the 5a and type I AFP genes share substantial sequence identity in the 5¢ and 3¢ untranslated regions of their genes. Thus, the argument for the hyperactive 17 kDa AFPs being homologous to the type I AFPs is strong even though it is indirect and relies on the 5a gene as a common link. That there are no small plasma AFPs in the American plaice argues that they were probably never developed in this fish rather than that they were selec- tively lost. The gene family coding for type I AFPs in winter flounder plasma has 30–40 members, many of which are organized in tandem head-to-tail arrays, which are a hallmark of gene amplification resulting from strong selective pressure [25]. It seems unlikely that such a robust gene family, which encodes func- tionally important proteins, would be completely lost or silenced when it is very highly expressed in closely related species. If this is the case, then the larger AFP could be the progenitor of the small type I AFPs. The presence of larger, highly active AFPs in the plasma of both winter and yellowtail flounder that are fully capable of protecting the fish from freezing below the freezing point of seawater calls into question the functional significance of the small type I AFP in these species. Considering the levels of these AFPs in the plasma it seems unlikely that they are excess to the needs of the fish. In addition, the abundance of the skin-type AFP in epithelial tissues also attests to the argument that these small AFPs are important to the survival of winter flounder and other species in subzero ice-laden waters [26,27]. Although the information that we have on the num- ber of species that possess the larger AFP is limited, it is evident that the two species (winter flounder, yellow- tail flounder) that have both the large and the small AFP normally reside in water that is considerably shal- lower than that inhabited by the American plaice [12]. Thus they are much more likely to be exposed to ice. The presence of the small skin-type AFP in the epithe- lial tissues of winter flounder suggests that they are required to protect the cells and tissues that would come into direct contact with ice [27,28]. The presence of skin-type AFP in a variety of species inhabiting the relatively shallow waters along the coast of Newfound- land during the winter makes a case for their physiolo- gical significance [29–31]. The larger 5a-like AFP produced by winter flounder is a dimer of > 30 kDa and behaves as a much larger protein during size-exclusion chromatography. There- fore it may not be capable of penetrating or diffusing readily into poorly vascularized tissues such as the skin epithelia. One other point worthy of mention in this regard is the freeze protection of urine. AFPs have been found in the urine of all AFP producing species that possess functional glomeruli (winter flounder, sea raven (Hemitripterus americanus), Atlantic cod (Gadus morhua) and ocean pout (Macrozoarces americanus) [32]. In the case of the winter flounder the AFP S. Y. Gauthier et al. Hyperactive antifreeze protein in fish FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS 4445 isolated from urine were of the small helical variety. The problems associated with freeze protection of extracellular fluids such as urine, and external epithelia could have been the impetus for the evolution of the smaller AFP. The significance of this report is three-fold: (a) large hyperactive AFPs are found in at least three genera of righteye flounder; (b) because only two of these three species have the smaller type I AFPs, it raises the pos- sibility that the type I AFPs have been derived from the large AFPs; and (c) if there are other flounder spe- cies which have been scored as AFP-negative because they do not have the type I AFPs they should be re-examined for the presence of the hyperactive, thermolabile large AFP. Experimental procedures Winter flounder (Pseudopleuronectes americanus) and yellow- tail flounder (Limanda ferrugenia) were collected from New- foundland coastal waters by commercial fishers and SCUBA divers and transported live to aquaria where they were main- tained in sea water until blood was sampled. American plaice (Hippoglossoides platessoides) were collected from the St. Lawrence estuary by otter trawl, transported live to St. Jolie, PQ where they were blood sampled. Blood samples were collected from a caudal blood vessel using 21 or 23-gauge syringe needles, transferred to heparinized test tubes and cen- trifuged to remove the blood cells. The resulting plasma was stored frozen at )20 °C prior to analysis. All measures were taken to minimize pain and discomfort during animal experiments. Guidelines followed were those of the Canadian Council on Animal Care (CCAC). Purification procedures The purification of a 17 kDa AFP from winter flounder has been described previously [13,14] Briefly, plasma was fractionated by size-exclusion chromatography, fractions containing the 17 kDa AFP were pooled and subjected to three successive rounds of ice affinity purification (IAP) [15]. The few remaining contaminants in the ice fraction were removed by batch adsorption to DEAE-Sephacel in 50 mm ammonium bicarbonate buffer (adjusted to pH 7.0 with HCl immediately before the chromatography). The 17 kDa AFP did not bind to the anion exchanger and was recovered by filtering the resin into a column. All steps were performed at or below 4 °C. The final product was examined for purity by SDS ⁄ PAGE and mass spectro- metry. Yellowtail flounder antifreeze proteins were partially purified by two rounds of ice affinity purification followed by the DEAE-Sephacel step. Frozen plasma was thawed and cleared by centrifugation. An aliquot (1.5 mL) from an individual fish was diluted with 30 mL of cold 20 mm ammonium bicarbonate (pH 7.9). Growth of an ice hemi- sphere in the plasma solution was initiated from a cold- finger at a temperature of )0.5 °C and the ice was grown to two-thirds of the sample volume (20 mL) by gradually lowering the temperature to )2.0 °C over 18 h. The ice fraction was then removed and melted, adjusted to 20 mm ammonium bicarbonate and a second round of IAP was performed prior to the removal of impurities using DEAE- Sephacel. Blood plasma from American plaice (pooled from several fish) was initially purified by three consecutive size-exclusion chromatography steps. An aliquot (3 mL) of plasma was dilu- ted with an equal volume of running buffer and loaded onto a 0.5 L (2.6 · 100 cm) Sephadex G-75 size-exclusion column at 4 °C and fractionated at a flow rate of 0.7 mLÆmin )1 with 100 mm ammonium bicarbonate (pH 7.9) as the running buffer. The fractions (5 mL) exhibiting TH activity were pooled, lyophilized, resuspended in running buffer (6 mL) and reapplied to the same column. The proteins were eluted as above. Active fractions were pooled and lyophilized. This pooled material was resuspended in 100 mm ammonium bicarbonate (pH 7.9) running buffer (0.2 mL) and loaded on a FPLC Superose-12 column (Pfizer, New York, NY, USA; 1.0 · 30 cm) at room temperature and eluted at 0.5 mLÆmin )1 . The Superose-12 column was calibrated with standard proteins of known molecular mass (Fig. 1 legend). To avoid the chromatography step carried out at room temperature, an alternative purification procedure for ApA- FP was developed. The plasma was fractionated by one cycle of Sephadex G-75 size-exclusion chromatography as described above. AFP was further enriched from the TH-active fractions [diluted to 42.5 mL with 100 mm ammonium bicarbonate (pH 7.9)] by one round of IAP using a cooling regime of )0.6 °Cto)3.6 °C in 24 h. Con- taminants were removed using DEAE-Sephacel as described above. Dilute samples of AFP were concentrated by retent- ion in an Amicon (Danvers, MA, USA) Ultra-15 centrifu- gal filter device (5 kDa molecular mass cut-off) at 4 °C. Amino acid analysis and N-terminal sequencing by Edman degradation The protein corresponding to the  17 kDa AFP in all three righteye flounders was partially or completely purified as described above and then electrophoresed on SDS ⁄ PAGE. After transferring the protein band(s) onto poly(vinylidene difluoride) membrane using 10 mm CAPS, 10% (v ⁄ v) methanol as transfer buffer (pH 11.0), the region corresponding to the 17 kDa band was visualized using Coomassie Blue R-250, cut out of the membrane and sent for N-terminal sequencing by Edman degradation (Advanced Protein Technology Centre, Hospital for Sick Children, Toronto, ON). Hyperactive antifreeze protein in fish S. Y. Gauthier et al. 4446 FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS Amino acid analyses were performed by the Advanced Protein Technology Centre, Toronto on the AFPs from winter flounder and American plaice after they had been purified to homogeneity following the DEAE-Sephacel step. These analyses were used to determine both the composi- tion and concentration of these proteins. Amino acid ana- lysis of the yellowtail flounder AFP was done directly on the  17 kDa band blotted from the SDS ⁄ PAGE gel. Mass spectrometry The mass of the hyperactive winter flounder AFP was determined previously [13]. The masses of the American plaice and yellowtail flounder AFPs were determined by MALDI-TOF mass spectrometry using protein samples taken following the DEAE-Sephacel purification step. The ApAFP sample was analyzed with a Micromass (Waters Ltd., Mississauga, Canada) Q-TOF Ultima instrument using a matrix of 5 mgÆmL )1 a-cyano-4-hydroxycinnamic acid, 0.1% trifluoroacetic acid in 70% acetonitrile. The mass of the yellowtail flounder protein was determined with a Voy- ager DE Pro spectrometer (Applied Biosystems, Foster City, CA, USA) in linear mode with a matrix of 10 mgÆmL )1 sin- apinic acid. Circular dichroism spectroscopy CD spectra of ApAFP were collected using an Olis Rapid Scanning Monochromator with a digital subtractive method CD module (Olis, Bogart, Georgia) and a 1 mm quartz cuvette (Hellma 121-QS). CD spectra were collected at 0.69 nm intervals between 260 and 180 nm at a scanning rate (nmÆmin )1 ) determined by the olis software as a func- tion of the signal intensity. The temperature of the cuvette holder was monitored and controlled by a circulating waterbath. An aliquot of the purified ApAFP was dialyzed exten- sively against 10 m m sodium phosphate (pH 7.0) at 4 ° C, and the baseline circular dichroism of this dialysis buffer was subtracted from the subsequent protein spectra. The concentration of AFP in this sample was estimated by amino acid analysis to be  3 lm . Seven spectra were col- lected from this protein sample near 4 °C and averaged. The temperature was then slowly increased to 75 °C (over 14.5 h) and spectra were collected continuously. The spec- tra collected within temperature ranges of 4–10 C° were averaged (4–8, 8–12, 12–16, 16–20, 20–24, 24–30, 30–40, 40–50, 50–60, and 60–75 °C). The measured ellipticity (mil- lidegrees) at each wavelength was converted to mean residue ellipticity (degÆcm )2 Ædmol )1 ) using the total concen- tration of all residues determined by amino acid analysis (0.70 mm). This value corresponds closely to that deter- mined using the estimated protein concentration and num- ber of amino acids in the protein, which were both predicted from the amino acid composition and molecular mass. The components of the secondary structure of the protein were deconvoluted from the CD spectrum (180– 260 nm) using the neural network-based software cdnn version 2.1 (Gerald Bo ¨ hm, Institut fu ¨ r Biotechnologie, Martin-Luther-Universita ¨ t Halle-Wittenberg, Germany) [33], trained on the ‘simple’ set of proteins. In a separate experiment melting and renaturation curves were examined. A sample of protein ( 4.2 lm) was dia- lyzed against 10 mm sodium phosphate (pH 7.0) and, as above, the dialysis buffer was used to establish the baseline. Circular dichroism of the sample was measured using an AVIV 62ADS CD spectrometer (Lakewood, NJ) and a quartz cuvette of 1 mm pathlength (Hellma 110-QS). The temperature of the cuvette was controlled by a Peltier device. The temperature of the sample was increased from 4to30°Cat1C° intervals every 2 min. Following equili- bration at each temperature, the circular dichroism at 222 nm of the sample was measured. As described above, the measured ellipticity was converted to mean residue ellipticity using a concentration determined by amino acid analysis. To investigate the reversibility of the helix to coil trans- ition that occurs between 7 and 12 °C, the sample was cooled from 30 to 4 °C. Because there was little change in the CD signal upon warming from 15 to 30 °C, the sample was rapidly cooled to 15 °C before beginning collection of CD data. The temperature was then lowered at 1 C° inter- vals and at each temperature the ellipticity at 222 nm was monitored until it appeared to stabilize. This required 10– 15 min for temperatures below 12 °C. When the CD signal appeared to stabilize, it was measured for 2 min and aver- aged before lowering the temperature an additional degree. To determine whether the protein would continue to rena- ture at a slow rate not detectable on a time scale of min- utes, the sample was stored at 4 °C for 40 h and the CD was measured again. Antifreeze assays Thermal hysteresis was measured as previously described [34]. Ice crystal images were digitally collected by a Nikon CoolPix 4500 camera mounted on a Leitz dialux 22 micro- scope with a Leitz Wetzlar 160 ⁄ –EF10⁄ 0.25 objective (Oberkochen, Germany). Acknowledgements This work was funded by grants from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council (NSERC) to P.L.D. and G.L.F., respectively. P.L.D. holds a Canada Research Chair in Protein Engineering. C.B.M. was supported by an Ontario Graduate Schol- arship. The authors would like to thank Madonna S. Y. Gauthier et al. Hyperactive antifreeze protein in fish FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS 4447 King for the shipment of fish plasma, Kim Munro and the Protein Function Discovery Facility at Queen’s University for circular dichroism spectroscopy, and the Alberta Peptide Institute, Edmonton, AB and the Advanced Protein Technology Centre at the Hospital for Sick Children, Toronto, ON for amino acid analysis and N-terminal sequencing. We also thank Avi Chakrabartty and the Ontario Cancer Institute for access to their CD machine. References 1 Raymond JA & DeVries AL (1977) Adsorption inhibi- tion as a mechanism of freezing resistance in polar fishes. Proc Natl Acad Sci USA 74, 2589–2593. 2 Fletcher GL, Kao MH & Fourney RM (1986) Anti- freeze peptides confer freezing resistance to fish. Can J Zool 64, 1897–1901. 3 Scholander PF, Vandam L, Kanwisher JW, Hammel HT & Gordon MS (1957) Supercooling and osmoregu- lation in Arctic fish. J Cell Comp Physiol 49, 5–24. 4 Scott GK, Fletcher GL & Davies PL (1986) Fish anti- freeze proteins: recent gene evolution. Can J Fish Aquat Sci 43, 1028–1034. 5 Fletcher GL, Hew CL & Davies PL (2001) Antifreeze proteins of teleost fishes. Annu Rev Physiol 63, 359–390. 6 Duman JG & DeVries AL (1974) Freezing resistance in winter flounder. Nature 274, 237–238. 7 Scott GK, Davies PL, Kao MH & Fletcher GL (1988) Differential amplification of antifreeze protein genes in the pleuronectinae. J Mol Evol 27, 29–35. 8 Knight CA, Cheng CC & DeVries AL (1991) Adsorp- tion of alpha-helical antifreeze peptides on specific ice crystal surface planes. Biophys J 59, 409–418. 9 Yang DS, Sax M, Chakrabartty A & Hew CL (1988) Crystal structure of an antifreeze polypeptide and its mechanistic implications. Nature 333, 232–237. 10 Sicheri F & Yang DS (1995) Ice-binding structure and mechanism of an antifreeze protein from winter floun- der. Nature 375, 427–431. 11 Baardsnes J, Kondejewski LH, Hodges RS, Chao H, Kay C & Davies PL (1999) New ice-binding face for type I antifreeze protein. FEBS Lett 463, 87–91. 12 Goddard SV & Fletcher GL (2002) Physiological Ecol- ogy of Antifreeze Proteins – a Northern Perspective. In Molecular Aspects of Fish and Marine Biology, Vol. 1: Fish Antifreeze Proteins. (Ewart KV & Hew CL, eds), pp. 17–60. World Scientific, Singapore. 13 Marshall CB, Fletcher GL & Davies PL (2004) Hyper- active antifreeze protein in a fish. Nature 429, 153. 14 Marshall CB, Chakrabartty A & Davies PL (2005) Hyperactive antifreeze protein from winter flounder is a very long, rod-like dimer of alpha-helices. J Biol Chem 280, 17920–17929. 15 Kuiper MJ, Lankin C, Gauthier SY, Walker VK & Davies PL (2003) Purification of antifreeze proteins by adsorption to ice. Biochem Biophys Res Commun 300, 645–648. 16 Davies PL & Gauthier SY (1992) Antifreeze protein pseudogenes. Gene 112, 171–178. 17 Hew CL, Wang NC, Yan S, Cai H, Sclater A & Fletcher GL (1986) Biosynthesis of antifreeze polypep- tides in the winter flounder. Characterization and seaso- nal occurrence of precursor polypeptides. Eur J Biochem 160, 267–272. 18 Wallimann P, Kennedy RJ, Miller JS, Shalongo W & Kemp DS (2003) Dual wavelength parametric test of two-state models for circular dichroism spectra of helical polypeptides: anomalous dichroic properties of alanine- rich peptides. J Am Chem Soc 125, 1203–1220. 19 Gronwald W, Chao H, Reddy DV, Davies PL, Sykes BD & Sonnichsen FD (1996) NMR characterization of side chain flexibility and backbone structure in the type antifreeze protein at near freezing temperatures. Biochemistry 35, 16698–16704. 20 Houston ME Jr, Chao H, Hodges RS, Sykes BD, Kay CM, Sonnichsen FD, Loewen MC & Davies PL (1998) Binding of an oligopeptide to a specific plane of ice. J Biol Chem 273, 11714–11718. 21 Scott GK, Davies PL, Shears MA & Fletcher GL (1987) Structural variations in the alanine-rich antifreeze proteins of the pleuronectinae. Eur J Biochem 168, 629– 633. 22 Chao H, Houston ME Jr, Hodges RS, Kay CM, Sykes BD, Loewen MC, Davies PL & Sonnichsen FD (1997) A diminished role for hydrogen bonds in antifreeze pro- tein binding to ice. Biochemistry 36, 14652–14660. 23 Hew CL, Wang NC, Joshi S, Fletcher GL, Scott GK, Hayes PH, Buettner B & Davies PL (1988) Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus. J Biol Chem 263, 12049–12055. 24 Davies PL, Hough C, Scott GK, Ng N, White BN & Hew CL (1984) Antifreeze protein genes of the winter flounder. J Biol Chem 259, 9241–9247. 25 Scott GK, Hew CL & Davies PL (1985) Antifreeze pro- tein genes are tandemly linked and clustered in the gen- ome of the winter flounder. Proc Natl Acad Sci USA 82, 2613–2617. 26 Gong Z, Ewart KV, Hu Z, Fletcher GL & Hew CL (1996) Skin antifreeze protein genes of the winter floun- der, Pleuronectes americanus, encode distinct and active polypeptides without the secretory signal and prose- quences. J Biol Chem 271, 4106–4112. 27 Murray HM, Hew CL, Kao KR & Fletcher GL (2002) Localization of cells from the winter flounder gill expressing a skin-type antifreeze protein gene. Can J Zool 80, 110–119. Hyperactive antifreeze protein in fish S. Y. Gauthier et al. 4448 FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS [...]... of skin-type antifreeze protein in winter flounder (Pseudopleuronectes americanus) epidermis following metamorphosis J Morphol 257, 78–86 29 Evans RP & Fletcher GL (2004) Isolation and purification of antifreeze proteins from skin tissues of snailfish, cunner and sea raven Biochim Biophys Acta 1700, 209–217 30 Low WK, Miao M, Ewart KV, Yang DS, Fletcher GL & Hew CL (1998) Skin-type antifreeze protein from... from the shorthorn sculpin, Myoxocephalus scorpius Expression and characterization of a Mr 9,700 recombinant protein J Biol Chem 273, 23098–23103 FEBS Journal 272 (2005) 4439–4449 ª 2005 FEBS Hyperactive antifreeze protein in fish 31 Low WK, Lin Q, Stathakis C, Miao M, Fletcher GL & Hew CL (2001) Isolation and characterization of skintype, type I antifreeze polypeptides from the longhorn sculpin, Myoxocephalus... octodecemspinosus J Biol Chem 276, 11582–11589 32 Fletcher GL, King MJ, Kao MH & Shears MA (1989) Antifreeze proteins in the urine of marine fish Fish Physiol Biochem 6, 121–127 33 Bohm G, Muhr R & Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks Protein Eng 5, 191–195 34 Chakrabartty A & Hew CL (1991) The effect of enhanced alpha-helicity on the activity... Quantitative analysis of protein far UV circular dichroism spectra by neural networks Protein Eng 5, 191–195 34 Chakrabartty A & Hew CL (1991) The effect of enhanced alpha-helicity on the activity of a winter flounder antifreeze polypeptide Eur J Biochem 202, 1057–1063 4449 . Hyperactive antifreeze protein in flounder species The sole freeze protectant in American plaice Sherry Y. Gauthier 1 , Christopher. is the only detectable antifreeze protein in the blood plasma of the American plaice. Because this species appears to lack the smaller type I antifreeze proteins,