Tài liệu Báo cáo khoa học: Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner ppt

Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner Yoshiyuki Nishimiya 1 , Ryoko Sato 1 , Manabu Takamichi 2 , Ai Miura 1 and Sakae Tsuda 1,2 1 Functional Protein Research Group, Research Institute of Genome-based Biofactory (RIGB), National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Japan 2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan Antifreeze protein (AFP) possesses the unique ability to bind to the surface of ice crystals, which permits growth of ice at limited open spaces on the surface, leading to the formation of numerous convex ice surfa- ces between the bound AFPs [1]. The growing ice sur- face becomes energetically unfavorable for further absorption of water molecules proportionately with the surface curvature, leading to termination of ice growth (Kelvin effect) [2]. This AFP-induced inhibition of ice crystal growth can be detected macroscopically as a depression in the freezing temperature (T f ) of the solution without alteration of the melting temperature (T m )(|T f ) T m |). This is generally termed thermal hys- teresis (TH). AFPs categorized as type III ( 7 kDa) have been identified in blood serum of fish living in polar sea- water with a year-round temperature of  )1.8 °C: Arctic and Antarctic eelpouts (Austrolycicthys brachy- cephalus, Lycodes polaris, and Lycodichthys dearborni), Atlantic ocean pout (Macrozoarces americanus), and Atlantic wolffish (Anarhichas lupus) [3–6]. Type III AFP forms a globular shape characterized by internal Keywords co-operative effect; Notched-fin eelpout; type III antifreeze protein Correspondence S. Tsuda, Functional Protein Research Group, Research Institute of Genome-based Biofactory (RIGB), National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan Fax: +81 11 857 8983 Tel: +81 11 857 8912 E-mail: sakae.tsuda@aist.go.jp Note The nucleotide and protein sequences reported here have been deposited in the DDBJ database under the accession numbers AB188389–AB188401. (Received 27 August 2004, revised 9 November 2004, accepted 17 November 2004) doi:10.1111/j.1742-4658.2004.04490.x We found that Notched-fin eelpout, which lives off the north east coast of Japan, expresses an antifreeze protein (AFP). The liver of this fish contains DNAs that encode at least 13 type III AFP isoforms (denoted nfeAFPs). The primary sequences of the nfeAFP isoforms were categor- ized into SP- and QAE-sephadex binding groups, and the latter were fur- ther divided into two subgroups, QAE1 and QAE2 groups. Ice crystals observed in HPLC-pure nfeAFP fractions are bipyramidal in shape with different ratios of c and a axes, suggesting that all the isoforms are able to bind ice. We expressed five recombinant isoforms of nfeAFP and ana- lyzed the thermal hysteresis (TH) activity of each as a function of pro- tein concentration. We also examined the change in activity on mixing the isoforms. TH was estimated to be 0.60 °C for the QAE1 isoform, 0.11 °C for QAE2, and almost zero for the SP isoforms when the con- centrations of these isoforms was standardized to 1.0 mm. Significantly, the TH activity of the SP isoforms showed concentration dependence in the presence of 0.2 mm QAE1, indicating that the less active SP isoform becomes ‘active’ when a small amount of QAE1 is added. In contrast, it does not become active on the addition of another SP isoform. These results suggest that the SP and QAE isoforms of type III AFP have dif- ferent levels of TH activity, and they accomplish the antifreeze function in a co-operative manner. Abbreviations AFGP, antifreeze glycoprotein; AFP, antifreeze protein; nfeAFP, type III AFP from Notched-fin eelpout; TH, thermal hysteresis. 482 FEBS Journal 272 (2005) 482–492 ª 2004 FEBS twofold symmetry of the ‘pretzel’ fold [7–14], which provides a markedly flat amphipathic ice-binding surface contributed by residues 9–10, 13–16, 18–21, 41, 42, 44 and 61 [15–18]. A large database of amino acid sequences is available for the various isoforms of type III AFP. The sequences of at least 12 isoforms (denoted HPLC1–12) have been determined for Macrozoarces americanus, which were originally categ- orized into QAE-Sephadex-binding and SP-Sephadex- binding groups [19]. The best-characterized AFP, HPLC12, is the only a member of the QAE group, all others (HPLC1–11) belonging to the SP group. The QAE and SP isoforms share only 55% identity among their primary sequences, although there is 90% identity among the SP isoforms [20]. Immunological cross-reac- tivity is also different between the QAE and SP iso- forms [5]. Post-translational modification has been reported only for the SP isoforms [19]. Of the residues that form the ice-binding surface, the hydrophilic ones are almost uniformly conserved in the QAE and SP isoforms, whereas the hydrophobic residues tend to be changeable. The same has been found for the AB1 and AB2 isoforms from Austrolycicthys brachycephalus [3]. They share 83% sequential identity with the same hydrophilic residues at the ice-binding surface, whereas the hydrophobic residues Pro19 and Ala20 located on the surface of AB1 are replaced with Ile19 and Val20 in AB2, respectively. At a concentration of 20 mgÆmL )1 , TH activity of AB1 is 1.27 °C, which is slightly higher than 1.17 °C of AB2. Lycodichthys dearborni also has three major AFPs (RD1, RD2, and RD3) [21]. RD3 is an exceptional isoform which com- prises two type III AFP domains connected in tandem through a nine-residue linker. The sequential identity between RD1 and RD2 is 94%, and the former shares 98% and 85% identities with the N-terminal and C-terminal domains of RD3, respectively (for RD2, the corresponding identities are 94% and 77%, respectively). For RD1 and RD2, the amino acid replacements of the hydrophobic residues (20th and 41st) have been identified for the ice-binding surface. At a concentration of 10 mgÆmL )1 , the TH activities of RD1 and RD2 are almost identical (0.95 °C and 0.90 °C, respectively), whereas RD3 has a slightly lower activity of 0.81 °C. Overall, the type III AFP isoforms differ in their hydrophobic residues but not significantly in their hydrophilic residues with regard to the ice-binding surface. One may speculate that these differences may differentiate the antifreeze func- tions of the isoforms. However, not much is known about the relationship between TH activity and the isoforms, especially the existence of a co-operative effect of the isoforms in the QAE and SP groups. We recently found that a significant amount of type III AFP can be purified from the minced muscles of Notched-fin eelpout ( Zoarces elongatus Kner), which lives off the north east coast of Japan ( 40° of lati- tude) where the temperature of the seawater is )1.7 °C in mid-winter. This fish naturally expresses at least 13 AFP isoforms (nfeAFPs). The primary sequences of two major species were examined using a protein se- quencer, and 13 sequences were independently deter- mined by analysis of cDNA sequences using the mRNA library constructed for the fresh liver of this fish. By comparing all of the identified sequences of nfeAFPs with those of ordinary type III AFP hetero- geneity [19], 13 isoforms, denoted nfeAFP1–13, were categorized into SP and QAE groups. The latter were further divided into QAE1 and QAE2 groups which are mainly distinguished by 10 characteristic residues. The QAE1 isoforms exhibit high sequence similarity to a well-known QAE isoform, HPLC12, from ocean pout. Here we expressed five recombinant isoforms, nfeAFP2 and nfeAFP6 (SP group), nfeAFP8 (QAE1 group), nfeAFP11 and nfeAFP13 (QAE2 group) so as to compare the TH activity between the groups. TH activity was also measured for a mixture of the SP and QAE isoforms to examine their co-operative effect on antifreeze activity. Results Preparation and sequence analysis of nfeAFPs Figure 1 shows the elution profile of the soluble pro- teins in the muscle homogenate of Notched-fin eelpout on the cation-exchange column. The mixture of Fig. 1. Elution profile obtained for the muscle homogenate of Notched-fin eelpout by cation-exchange chromatography (High-S column). A linear gradient (dotted line) from 0 to 500 m M NaCl at a flow rate of 1 m LÆmin )1 was used to elute the fractions containing the isoforms of nfeAFP. Y. Nishimiya et al. Co-operative effect of type III AFP isoforms FEBS Journal 272 (2005) 482–492 ª 2004 FEBS 483 nfeAFP isoforms was eluted by application of a con- centration gradient of NaCl ( 50–250 mm). Their activity was confirmed by photomicroscopic observa- tion of the bipyramidal ice crystal. The mixture of nfeAFPs migrated as a  4.5-kDa band on SDS ⁄ PAGE, which is smaller than the actual molecular mass ( 7 kDa), as previously observed [5,22]. The mixture of the two fractionated nfeAFPs was resolved into six major and eight minor peaks labeled 1–14 in Fig. 2A by RP-HPLC using a C 18 reverse-phase col- umn. The molecular mass of  6600 was estimated to the peaks 1–10, and  7000 to the peaks 11–14 by MALDI-TOF MS. An elongated bipyramidal ice crys- tal in the c-axis direction was observed (Fig. 2B) for peaks 1–10 eluted in the 41–54% concentration range of acetonitrile (the hexagonal shape observed for peak 1 is attributable to the low protein concentration). In contrast, a thick bipyramidal ice crystal was detected for peaks 11–14 eluted in the range above 54% aceto- nitrile. We analysed the amino acid sequence for peak 2 in Fig. 2A (later assigned to nfeAFP6) and its cleaved fragments using a conventional sequence analyzer. As shown in Fig. 3, the amino acid sequence of residues 1–56 was determined (denoted A1) for a nondigested component of the peak. Figure 3 also shows the sequence determinations of five fragments of peak 2 (denoted N1–N5) cleaved by asparaginyl endopeptidase and five fragments of the peak cleaved by TPCK tryp- sin (T1–T5). By comparing the cleaved sequences with each other, the N1 and T1 fragments were assigned to the N-terminal sequence, and N5 and T5 to the C-ter- minal sequence. The C-terminus was assigned to Ala as it would not be cleaved by the proteases used. The other sequential overlaps between A1, N2, N3, N4, T2, T3, and T4 allowed us to identify that A1 consists of 56 of the 64 residues of this isoform, in which the unknown 35th and 48th residues were assigned to Glu and Val, respectively. The information on the com- plete sequence analyzed for peak 2 allowed us to iden- tify that the isoform belongs to the type III AFP the N-terminus of which is unblocked, probably the SP group isoform identified from ocean pout [19]. An incomplete 62-residue sequence determined for a non- digested component of peak 8 in Fig. 2A is also shown in Fig. 3 (labeled A2). It should be noted that both A1 and A2 have Gly at the N-terminus, which is similarly identified in the SP isoforms from ocean pout. cDNA cloning and sequence alignment of nfeAFPs The complete amino acid sequences of the 13 isoforms of nfeAFP were determined based on the cDNA A B Fig. 2. (A) HPLC purification of the isoforms of type III AFP from Notched-fin eelpout. The peaks containing the HPLC-pure isoforms are labeled 1–14. The amino acid sequence was analysed for peaks 2 and 8 (Figs 3 and 4). (B) Photomicrographs of the single ice crystal observed for each HPLC-pure isoform dissolved in 0.1 M NH 4 HCO 3 (pH 7.9). Co-operative effect of type III AFP isoforms Y. Nishimiya et al. 484 FEBS Journal 272 (2005) 482–492 ª 2004 FEBS library as described in Experimental procedures. The N-terminal residue of the 13 intact AFPs was determined by reference to the ordinary type III AFP sequences and the identified signal sequences of A1 and A2: i.e. MKSVILTGLFFVLLCVDHMSSA for nfeAFP11 and 12 and MKSVILTGLLFVLLCVD HMSSA for nfeAFP1–10, 13. The signal sequence of nfeAFP2 was only partially identified from cDNA. The sequence A1 is identical with the 1st 65 residues of nfeAFP6 with the 66th lysine residue at the C-ter- minus, which is presumably removed by the post- translational processing [19]. Although such processing can be assumed for nfeAFP1–5 and nfeAFP10 which have Lys at their C-termini, we assumed a Lys-con- taining sequence as the complete amino acid sequence of the isoforms, as no direct evidence has been obtained so far to indicate their deletion. For nfeAFP6, the effect of a C-terminal lysine on activity was examined, as described later. Interestingly, the sequence A2 is not identical with any of the nfeAFP sequences listed in Fig. 4, although it shows maximum similarity to nfeAFP2: Ala23, Ala35, and Met37 of A2 are substituted by Glu, Val, and Ala of nfeAFP2, respectively. Most of the isoforms are about 65 resi- dues in length; only nfeAFP13 consists of 70 residues. nfeAFP13 exceptionally contains two cysteines (Cys64 and Cys66), the sequence of which shows high similar- ity to an isoform denoted ‘genomic k3’ identified from ocean pout [20]. The presently identified 13 isoforms of nfeAFP do not exhibit 100% identity with any other reported sequences of type III AFP. Based on the sequence similarity to the known type III AFP isoforms from Atlantic ocean pout [20], the 13 isoforms of nfeAFP were categorized into SP and QAE groups as listed in Fig. 4, for which there is a typical difference in the 34th to 37th residues; therefore sequence gaps (–) were introduced into positions 37 and 34 for the SP and QAE groups, respectively. A1, A2, and nfeAFP1–6 were categorized into the SP group and nfeAFP7–13 into the QAE group. For clar- ity, Gly1 of the SP group is defined as the 2nd residue in the present study. We further divided the QAE iso- forms into two subgroups, QAE1 (nfeAFP7–10) and QAE2 (nfeAFP11–13), which are distinguished by 10 characteristic residues colored blue and green in Fig. 4. The sequence identity within the 13 isoforms is  48%. The identity is 77% when compared within the SP iso- forms, 76% within the QAE1 isoforms, and 91% within the QAE2 isoforms, respectively. With regard to the putative ice-binding residues indicated with asterisks (*) in Fig. 4, the 42nd residue is different between the SP and QAE groups. In addition, Gln9, Leu19, Val20 and Val41 in the QAE1 group are replaced by Val9, Val19, Gly20 and Ile41, respectively, in the QAE2 group. It is worth noting that Gln9 is conserved in all known isoforms of type III AFP, except HPLC7 which contains Arg9 [20]. Overall, the hydrophilic residues are mostly conserved among the nfeAFP isoforms for the ice-binding residues (for example, Gln9, Asn14, Thr15, Thr18, and Gln44). However, significant amino acid replacements are iden- tified for the hydrophobic residues located at the 13th, 19th, 20th, and 41st positions. Antifreeze activity of recombinant nfeAFPs To examine the relationship between TH activity and sequence diversity of type III AFPs, the following five isoforms, listed in Fig. 4, were expressed and purified: nfeAFP2 (SP group); nfeAFP6, a major isoform of the muscle homogenate (SP group); nfeAFP8, the sequence of which is similar to HPLC12 (QAE1 group); nfeAFP11, a Val9-containing isoform (QAE2 group); nfeAFP13, the largest isoform containing Peak 2 1 10 20 30 40 50 A1 GESVVATQLIPINTALTPAMMEGKVTNPSGIPFAxMSQIVGKQVNTPxAKGQTLMP N1 GESVVATQLIPIN N2 TALTPAMMEGKVTNPSGIPFAxMSQIVGxQVN N3 TALTPAMMEGKVTN N4 PSGIPFAEMSQIVGxQVNTPVAxGQTL N5 MVKTYVPA T1 GESVVATQLIPINTALTPAMMEGK T2 VTNPSGIPFAEMSQ T3 QVNTPVAK T4 GQTLMPGMVK T5 TYVPA Peak 8 1 10 20 30 40 50 60 A2 GQSVVATQLIPMNTALTPAMMAGKVTNPSGIPFAEMSQIVGKQVNVIVAKGQTLMPDMVKTY Fig. 3. Amino acid sequences of the cleaved peptides obtained from the HPLC peaks. A1, 56 amino acid residues of peak 2 deter- mined by the sequencer; N1–N5, fragments of peak 2 cleaved by asparaginyl endopepti- dase; T1–T5, fragments of peak 2 cleaved by TPCK trypsin; A2, 62 amino acid residues of peak 8 determined by sequencer. Y. Nishimiya et al. Co-operative effect of type III AFP isoforms FEBS Journal 272 (2005) 482–492 ª 2004 FEBS 485 Val9 and two cysteines (Cys64 and Cys66) (QAE2 group). NfeAFP6 and nfeAFP8 exhibit the highest sequence identity with the isoforms HPLC6 and HPLC12 from M. americanus, respectively (the amino acid sequences of HPLC6 and HPLC12 are listed in Fig. 4). Again both nfeAFP2 and nfeAFP6 were expressed with C-terminal lysines. We also expressed nfeAFP6minusLys66 (nfeAFP6DLys; SP group) to examine the effect of the C-terminal lysine on activ- ity. For nfeAFP13, TH activity was determined in the presence and absence of reductant (dithiothreitol), as it can form multimers via intermolecular disulfide bridges (Fig. 5). Figure 6 shows the molar concentration dependence of TH activity for the six genetically produced iso- forms of nfeAFP examined using the Vogel osmo- meter. A nonlinear profile of TH activity typical of ordinary AFPs was identified for nfeAFP8 (QAE1 group), although its maximum activity ( 0.7 °C) was slightly lower than that reported for HPLC12, which was determined using the Clifton nanoliter osomome- ter [7]. A similar profile was detected for nfeAFP13 (QAE2 group) in the absence of reductant. The addi- tion of reductant significantly lowered the activity of nfeAFP13, indicating that the monomer is less active than when a small amount of multimers is present (Fig. 5). An extremely low level of TH activity was detected for another QAE2 isoform, nfeAFP11. We detected no appreciable TH activity for nfeAFP2, nfeAFP6, and nfeAFP6DLys (SP group). The lack of a significant difference between nfeAFP6 and nfeAFP6DLys suggests that a lysine at the C-terminus does not affect the ice-binding function, which is con- sistent with previous indications for ocean pout AFPs [19]. It is worth noting that nfeAFP2 and nfeAFP6 both form a folded structure, which was evidenced by a number of secondary shifts observed throughout the range of their 1 H-NMR spectra. TH activity at a con- centration of 1.0 mm was 0.60 °C for nfeAFP8 (QAE1 isoform), 0.44 °C (multimers) and 0.11 °C (monomer) for nfeAFP13 (QAE2 isoform), 0.02 °C for nfeAFP11 (QAE2 isoform), and almost zero for nfeAFPs 2 and 6 (SP isoforms). Formation of an elongated bipyramidal ice crystal was observed for the solutions of nfeAFP2 (0.3 mm) and nfeAFP6 (3.75 mm) at a cooling rate of Fig. 4. Alignment of amino acid of sequence of type III AFP identified from Notched-fin eelpout (nfeAFP). Spaces are introduced to optimize the alignment. The yellow color indicates the conserved residues of the nfeAFP isoforms. Ten characteristic residues colored blue and green distinguish the QAE1 and QAE2 groups. The putative ice-binding residues are indicated with asterisks. The sequences of HPLC6 and HPLC12 isoforms from M. americanus are also shown for comparison. Fig. 5. SDS ⁄ PAGE (16% gel) of a purified cysteine-containing QAE2 isoform of type III AFP from Notched-fin eelpout (nfeAFP13). Lane A, 0.3 m M nfeAFP13 in the presence of 10 mM of dithiothrei- tol (DTT); lane B, 0.3 m M nfeAFP13 in the absence of dithiothreitol. The protein standards (MW) are indicated on the left. The mono- mer of nfeAFP13 is the dominant species irrespective of the addi- tion of reductant. Co-operative effect of type III AFP isoforms Y. Nishimiya et al. 486 FEBS Journal 272 (2005) 482–492 ª 2004 FEBS 0.01–0.05 °CÆmin )1 , suggesting that these species have the ability to inhibit ice growth. We further examined whether the addition of a small amount (0.2 mm) of ‘active’ nfeAFP8 influences the TH activity of ‘less active’ nfeAFP6. Approxi- mately 0.10 °C of TH activity was detected (Fig. 6). As shown in Fig. 7, the TH activity of the less active nfeAFP6 shows clear concentration dependence in the presence of 0.2 mm nfeAFP8 (maximum TH ¼ 0.60 °C). A similar TH profile was obtained for nfeAFP6DLys in the presence of 0.2 mm of nfeAFP8. These data indicate that ‘less active’ AFP isoforms can exert a substantial level of antifreeze activity after the addition of a small amount of ‘active’ isoform. The less active nfeAFP6 does not, however, become active after the addition of another less active isoform, nfeAFP2 (Fig. 7). No significant difference was detec- ted between nfeAFP6 and nfeAFP6DLys, which con- firms that the C-terminal lysine does not directly participate in the antifreeze function. Discussion We have identified new isoforms of type III AFP from Notched-fin eelpout living off the Japanese coast at  40° of latitude. The isoforms were categorized into SP and QAE groups according to the known difference in residues 34–37. The QAE group was further divided into two subgroups distinguished by 10 characteristic residues (Fig. 4). A QAE1 isoform, nfeAFP8, exhibits the highest sequence identity with HPLC12, which was previously identified from Atlantic ocean pout (Ala24, Met30, Val35, Glu58, and Thr64 in nfeAFP8 are replaced by Ser24, Val30, Glu35, Asp58, and Pro64 in HPLC12). Therefore we categorized HPLC12 into the QAE1 group. It has been shown that HPLC12 is eluted last during HPLC [19], implying that the QAE isoform is more hydrophobic than the SP isoform. Although we did not complete the assignment of the HPLC peaks (Fig. 2) to the present isoforms, we detec- ted a difference in molecular mass between HPLC peaks 1–10 ( 6600 Da) and peaks 11–13 ( 7000 Da) (Fig. 2). Hence, it is highly likely that QAE isoforms of nfeAFP are eluted later in HPLC (Fig. 2), which agrees with the earlier elution of the SP isoforms (for example, peak 2, nfeAFP6). In addition, the bipyrami- dal ice crystals observed for the HPLC-pure samples labeled 11–14 have a low c ⁄ a axial ratio (i.e. they are thick in shape) compared with samples 2–10 of Fig. 2. A low c ⁄ a axial ratio has been suggested to be a sign of higher TH activity [8,18,23]. Hence, one can specu- late that samples 11–14 correspond to QAE isoforms of nfeAFP, whereas the others correspond to SP iso- forms, and the TH activity of the QAE isoform is higher than that of the SP isoform. Fig. 6. TH activity measured using an osmometer (model OM 802; Vogel) as a function of concentration (m M) of type III AFP iso- forms, nfeAFP2 (h), nfeAFP6 (r), nfeAFP6DLys (e), nfeAFP8 (·), nfeAFP11 (s), nfeAFP13 in the absence of dithiothreitol (n), and nfeAFP13 in the presence of dithiothreitol (m). The measure- ment was repeated three times using fresh samples, and mean values were plotted with error bars. Fig. 7. TH activity measured as a function of concentration (mM) of type III AFP isoforms: nfeAFP6 (m) and nfeAFP6DLys (s) in the presence of 0.2 m M nfeAFP8; nfeAFP6 (r) and nfeAFP6DLys (h)in the presence of 0.2 m M nfeAFP2. The measurements were repea- ted three times using fresh samples and mean values were plotted with error bars. Y. Nishimiya et al. Co-operative effect of type III AFP isoforms FEBS Journal 272 (2005) 482–492 ª 2004 FEBS 487 Although we identified ice-shaping activity for the SP isoforms of nfeAFP (Fig. 2), their TH activities were below the level of detection of the instrument used (Vogel osmometer) (Fig. 6). TH activity could also not be detected for 15Eklac, a 15-residue syn- thetic peptide corresponding to the 11-residue repeat- ing unit of a 36-residue type I AFP, using the Clifton nanoliter osmometer [24]. This minimized peptide forms a vertex-flat bipyramid of ice crystal, for which it was assumed imperfect inhibition of the crystal growth of f20  21g plane, a target ice surface of an intact type I AFP. Such an unsatisfactory level of ice growth inhibition leading to nondetection of TH activity is also assumed for the present SP isoform. Obviously, there are many candidates among the resi- dues that could offer such character to the SP iso- form. However, the residues located in the functional site (i.e. ice-binding region) are thought to be the prime candidates, as variants of type III AFP form a mostly identical 3D structure irrespective of the amino acid replacements [7–10,13,14]. As shown in Fig. 4 (*), replacements between the three isoforms are identified for the hydrophobic residues located at positions 9, 13, 19, 20, 41, and 42 of the putative ice- binding sequence. A clear difference between the SP and QAE isoforms is identified for the 42nd residue; Ser42 in QAE isoforms is replaced by Gly42 in SP isoforms. However, a mutant of HPLC12 (QAE1), S42G, has been reported to exhibit full TH activity [11], therefore the character of the SP isoform cannot be ascribed to Gly42 alone. It has also been reported for HPLC12 that replace- ment of a hydrophobic residue with a smaller aliphatic residue results in loss of TH activity; 23–28% loss was reported for the mutants L19A, V20A, and V41A. Furthermore, 55% loss of activity has been reported for the double mutant L10A⁄ I13A, and 75% loss for L19A ⁄ V41A [18]. Leu19 and Val20 of the QAE1 iso- form are replaced by Pro19 and Ala20 in the presently examined SP isoforms, nfeAFP2 and nfeAFP6. The replacement of these residues presumably alters the ice-binding character of the AFP isoform. A ‘semi- reversible’ ice-binding model for the kinetics of AFP- induced ice growth inhibition has been proposed [25,26], which includes the following adsorption steps of AFP: (a) attachment to the ice–water interface; (b) rearrangement of adsorbed molecules by diffu- sion, reorientation, and ⁄ or conformational change; (c) detachment from the interface. Again ice-shaping ability is suggested to be a charac- teristic of all isoforms of nfeAFP (Fig. 2). The hydro- phobic residues located at positions 9, 13, 19, 20, 41, and 42 are structured so as to surround the ice-binding surface in the 3D structure of type III AFP (PDB Code ¼ 1MSI). Hence, it can be speculated that a set of hydrophobic residues in an isoform differentiates the surface complementarity with the target plane of the ice crystal, which affects adsorption steps (b) and (c) espe- cially, resulting in a different level of TH activity. A clear concentration dependence of TH activity was observed for a QAE1 isoform (nfeAFP8) simi- larly to HPLC12, whereas it was below detectable level for the QAE2 isoforms (nfeAFP11 and nfeAFP13) (Fig. 6). It should be mentioned that Gln9, a highly conserved residue in the known type III AFPs, is replaced with Val9 in the QAE2 iso- forms. The Cys-containing QAE2 isoform, nfeAFP13, has the ability to form trimers and tetramers in the absence of reductant (dithiothreitol), although mono- mers are predominantly formed irrespective of the presence of reductant (Fig. 5). The difference in TH activity of nfeAFP13 in the presence and absence of dithiothreitol (Fig. 6) implies that the TH activity of the monomer (+ dithiothreitol) is enhanced approxi- mately threefold by the presence of a small amount of trimers and tetramers (– dithiothreitol). We previ- ously reported that an artificial multimer of type III AFP has considerably increased TH activity com- pared with the monomer [27]. In addition, for type I AFP isoforms from winter flounder consisting of 11 amino acid repeats, a longer isoform consisting of four repeats showed almost twice the activity of the ordinary three-repeat isoforms [28]. For b-helical AFP isoforms from spruce budworm, the activity of CfAFP-501 ( 12 kDa) with two additional loops is twofold higher than that of CfAFP-337 ( 9 kDa) [29]. Baardsnes et al. [30] recently reported that the increase in activity of the type III AFP multimer results from an increase in the size of the ice-binding surface. These results suggest that, although the monomer of nfeAFP13 does not exert substantial TH activity by itself, it is enhanced with the help of a small amount of multimers. A similar observation was reported for antifreeze glycoprotein (AFGP) from Antarctic cod, Pagothenia borchgrevinki, the molecular mass of which is in the range 2.6–33 kDa: the low TH activity of smaller sized AFGP ( 3 kDa) is markedly enhanced by the addition of the larger spe- cies ( 10.5 kDa) [31]. We found that TH activity of a SP isoform, nfeAFP6, is greatly enhanced by, and showed clear concentration dependence on, the addition of a small amount of a QAE1 isoform, nfeAFP8 (Fig. 7). This is similar to the case of the QAE2 isoform, nfeAFP13; the activity of its monomer was enhanced by the pres- ence of a small amount of the multimer. Although Co-operative effect of type III AFP isoforms Y. Nishimiya et al. 488 FEBS Journal 272 (2005) 482–492 ª 2004 FEBS there is no direct experimental evidence to explain the mechanism, one can assume the following co-operative ice-binding mechanism: (a) the ‘active’ AFP isoform (QAE1) firstly adsorbs to the ice crystal, which decrea- ses its growing speed and lowers the energy barrier to allow adsorption of the ‘less active’ isoform; (b) the less active isoform (SP or QAE2) can then adsorb to the ‘open space’ between the prebound AFPs of the ice crystal surface; (c) most of the nfeAFP isoforms adsorb to the growth-terminated ice crystal in the final state. This hypothesis is comparable to that of Bur- cham et al. [31]. They assumed that stabilization of the antifreeze action of small (weak) species of AFGP (AFGP6-8) by large (strong) species (AFGP1-5) pro- duces co-operative coverage of a seed ice crystal, thereby preventing further crystal growth. When we added a less active SP isoform (nfeAFP2) to another less active SP isoform (nfeAFP6) (Fig. 7), the co-operative ice binding did not occur, resulting in no substantial TH activity for the less active isoform. It is interesting to note that the less active SP iso- form (nfeAFP6) is the major AFP species produced in Notched-fin eelpout (Fig. 2). A similar observation was reported for AFGP; the concentration of the smal- ler sized AFGP in the serum of Antarctic cod is more than eightfold that of the larger AFGP components [32]. Ocean pout also produces many SP isoforms (11 species) compared with one QAE isoform [20]. Why is the less active SP isoform of type III AFP dominant in the fish serum? In winter, the level of expression of AFPs is maximized to  20–30 mgÆmL )1 in the serum [33]. The SP isoform is less hydrophobic, as evidenced by the present HPLC experiment (Fig. 2), and indeed is more soluble than the QAE isoform (data not shown). Hence, a plausible explanation for the sub- stantial content of the SP isoform is as follows: (a) the SP isoform is generated to reduce the hydrophobicity and improve the solubility of type III AFP isoforms as a whole at the expense of surface complementarity; (b) accordingly, the SP isoform is the species that cannot exert substantial TH activity by itself; (c) although the antifreeze activity of the SP isoform is low, it can exert TH activity with the help of the QAE isoform. The production of a number of AFP isoforms may be a strategy to retain AFPs in the serum at a sufficiently high concentration to prevent the serum from freezing. To summarize, we have succeeded in identifying 13 new isoforms of type III AFP from Notched-fin eel- pout, which were categorized into three groups: SP, QAE1, and QAE2. We detected a clear difference in TH activity between the isoforms, although ice-binding ability was detected for all of them. This was ascribed to differences in hydrophobic residues located in the ice-binding region. The less active SP isoform becomes active on addition of a small amount of the active QAE1 isoform, whereas it does not become active on addition of another less active SP isoform. These results suggest that isoforms of type III AFP co-opera- tively exert the antifreeze function. Experimental procedures Purification and sequence analysis of nfeAFPs Type III AFP was purified from the muscle of Notched-fin eelpout. After removal of the head and gut, the meat of the fish was homogenized with water using an electric mixer [tissue ⁄ water ratio (g) ¼ 1 : 1]. The homogenate was centri- fuged at 3000 g for 30 min, and the supernatant obtained dialyzed against 50 mm sodium acetate (pH 3.7) overnight at 4 °C. After removal of the precipitate formed during dialysis, the AFP-containing solution was loaded on to a high-S column (1.0 · 5.0 cm; Bio-Rad, Hercules, CA, USA), and the column-bound AFPs were eluted with a lin- ear NaCl gradient (0–0.5 m )in50mm sodium acetate buf- fer (pH 3.7). The fractions containing the isolated AFP were collected and further chromatographed by RP-HPLC using a C 18 reverse-phase column (TOSOH, Tokyo, Japan; TSKgel ODS-80Ts) with a linear gradient of 0–100% aceto- nitrile in 0.1% trifluoroacetic acid. For observation of ice- crystal morphology, the lyophilized powder of the AFP collected was dissolved in 0.1 m NH 4 HCO 3 . For amino acid sequence analysis, the AFP was digested with asparaginyl endopeptidase (TaKaRa, Shiga, Japan) at 37 °C for 24 h in 50 mm sodium acetate (pH 5.0) containing 10 mm dithio- threitol and 1 mm EDTA. Another digested fragment was obtained by incubation with TPCK trypsin (Pierce, Rock- ford, IL, USA) for 24 h at 37 °C in 0.1 m NH 4 HCO 3 (pH 7.9). These digested fragments were separated by RP-HPLC. Sequence analysis of the fragments and native AFP was carried out with an Applied Biosystem (Foster City, CA, USA) 491 protein sequencer. PCR amplification and cDNA sequencing of nfeAFP Fresh liver from a Notched-fin eelpout caught in the middle of the winter of 2001 was cut into 0.5-cm-thick slices, and soaked in RNA stabilization reagent (Qiagen, Hilden, Germany) overnight at )4 °C. After being frozen at )80 °C, 25 mg frozen liver was completely ground with liquid nitrogen using a mortar and pestle, and homogenized using a shredder spin column (Qiagen). Total RNA was then isolated from the liver using an RNeasy Protect kit (Qiagen). mRNA was purified from total RNA using the Oligotex-dT30 mRNA Purification kit (TaKaRa). A cDNA library was generated from 1.6 lg mRNA with the Y. Nishimiya et al. Co-operative effect of type III AFP isoforms FEBS Journal 272 (2005) 482–492 ª 2004 FEBS 489 ZAP-cDNA Synthesis kit (Stratagene, La Jolla, CA, USA). PCR was performed for a major cDNA consisting of 500 bp purified from the cDNA library using the templates of Ex-Taq DNA polymerase (TaKaRa), oligo-dT linker primer (5¢-GAGAGAACTAGTCTCGAGTTT-3¢), and the synthetic primer of the adapter sequence (5¢-TCGGG AATTCGGCACGAGG-3¢). The annealing sites of these primers were connected to 3 ¢-terminus and 5¢-terminus of cDNA. The PCR conditions are as follows: denaturing at 94 °C for 1 min, 2 cycles pre-extending at 94 °C for 1 min, extending at 56 °C and 72 °C for 1 min each, and 28 cycles extending at 94 °C, 50 °C, and 72 °C for 1 min each. The PCR products obtained were purified and ligated into pGEM-T Easy (Promega, Madison, WI, USA). The cloned DNAs encoding nfeAFP isoforms were sequenced using the ABI Prism Big dye terminator cycle sequencing kit and ABI 3100 genetic analyzer (Applied Biosystems). Expressions and purification of the five recombinant nfeAFPs The five DNA fragments encoding nfeAFP2, 6, 8, 11, and 13, from which the signal sequence was removed, were amplified by PCR using cloning plasmid vectors, and ligated into pET20b (Novagen) with the restriction enzymes, NdeI and XhoI. The plasmid-DNAs obtained were transformed into Esherichia coli strain BL21 (DE3), and the transformants were grown at 28 °C in Luria–Bertani medium supplemented with 100 lgÆmL )1 ampicillin, until cell growth reached the early stationary phase. To induce expression of the recombinant nfeAFPs, 0.5 mm isopropyl thio-b-d-galactoside was added to the medium, and the cul- tures were grown at 28 °C overnight. Most of the nfeAFPs were expressed as the soluble form, but nfeAFP13, which contains two cysteines (Cys64 and Cys66), was only expressed as the inclusion body. Hence, the nfeAFP13 sam- ple was prepared by the methods described in [15] with the following modifications: (a) the inclusion body was dis- solved in 100 mm Tris ⁄ HCl (pH 8.5) containing 6 m guani- dine hydrochloride and 10 mm 2-mercaptoethanol; (b) nfeAFP13 was then refolded with 50 mm K 2 HPO 4 ⁄ 100 mm NaCl ⁄ 10 mm 2-mercaptoethanol (pH 10.7). The nfeAFP isoforms obtained were dialyzed against 50 mm sodium acetate buffer (pH 3.7) and then purified by cation- exchange chromatography with a linear NaCl gradient (0–0.5 m)in50mm sodium acetate buffer (pH 3.7) (50 mm sodium citrate buffer, pH 2.9, was used for the purification of nfeAFP11). The fractions containing the purified nfeAFPs were stored and dialyzed against 0.1 m NH 4 HCO 3 (pH 7.9). The purity was checked by SDS ⁄ PAGE (16% gel) [34]. It should be noted that the purified nfeAFP13 appeared to form multimers via intermolecular disulfide bridges; nfeAFP forms a monomer in the presence of reductant (+ dithiothreitol), while its trimer and tetramer were also generated in the absence of reductant as shown in Fig. 5. Therefore, for the measurement of TH activity, nfeAFP13 was reduced for 12 h at 4 °C with 0.1 m NH 4 HCO 3 (pH 7.9) containing 10 mm dithiothreitol, and activity was measured on fresh samples. Measurement of ice crystal morphology and TH activity Ice-crystal morphology was observed using an in-house photomicroscope system consisting of a Leica DMLB 100 photomicroscope equipped with a Linkam LK600 (liquid nitrogen-type) temperature controller and a CCD camera. A droplet ( 0.5 lL) of the sample solution was frozen and then heated until a single ice crystal was observed sepa- rately in the solution by manipulation of the temperature controller. The change in morphology of a single ice crystal into a hexagonal bipyramid caused by the accumulation of AFP on the ice surfaces was then observed at a cooling rate of 0.05 °C per minute. The Clifton nanoliter osmometer (Clifton Technical Phys- ics, Hartford, NY, USA) is usually used to determine the ice-growth-initiation temperature, which is defined as the freezing point (T f ) of the AFP solution. However, this instru- ment is no longer available commercially, so we used the fol- lowing method with an alternative osmometer (model OM 802; Vogel): (a) 50 lL sample solution was placed in the cooling pot ()7 °C) of the instrument; (b) the frosty probe was put manually into the supercooled sample to initiate water freezing; (c) the increase in solution temperature caused by latent heat emission was monitored; (d) the plat- eau temperature was determined to be the T f point of the sample. All the nfeAFP samples were dissolved in 0.1 m NH 4 HCO 3 (pH 7.9) for the T f measurement. The melting temperature (T m ) of the AFP solutions was carefully deter- mined by monitoring the melting of an ice crystal on the photomicroscope stage, which was manipulated by the LK600 temperature controller. The measurement of T f and T m was repeated three times using fresh samples, and mean values were used for determination of TH activity (TH ¼|T m – T f |). It has been documented that the TH value determined using this osmometer is slightly lower than that determined using the Clifton nanoliter osmometer [13,27,30]. Acknowledgements We thank Dr Tamotsu Hoshino, Michiko Kiriaki, and Mineko Fjiwara for analysis of amino acid sequences, and Yumika Miura for analysis of DNA sequences. References 1 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. Co-operative effect of type III AFP isoforms Y. 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(1984) Analysis of antifreeze glycoproteins in fish serum Anal Biochem 139, 197–204 33 Fletcher GL Hew CL Li XM Haya K & Kao MH (1985) Year-round presence of high levels of plasma antifreeze peptides in a temperate fish, ocean pout (Macrozoarces americanus) Can J Zool 63, 488–493 492 Y Nishimiya et al 34 Laemmli U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage... bacteriophage T4 Nature 227, 680–684 Supplementary material The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4490/EJB4490sm.htm Figs S1–8 1H-NMR spectra of the recombinant isoforms of nfeAFP2, 6, and 13 (+ dithiothreitol) (S1– S3) and MALDI-TOF MS data for HPLC peaks 2, 6, 7, 8, and 13 of Fig 2 (S4–S8) FEBS Journal 272 (2005) 482–492 ª 2004 FEBS . Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner Yoshiyuki Nishimiya 1 ,. sequences of the 13 isoforms of nfeAFP were determined based on the cDNA A B Fig. 2. (A) HPLC purification of the isoforms of type III AFP from Notched-fin