Structural and functional characterization of a C-type lectin-like antifreeze protein from rainbow smelt ( Osmerus mordax ) John C. Achenbach 1, * and K. Vanya Ewart 2 1 Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada; 2 NRC Institute for Marine Biosciences, Halifax, Nova Scotia, Canada Antifreeze proteins (AFPs) are produced by several cold-water fish species. They de press physiological freezing temperatures by inhibiting growth of ice crystals and, in so doing, permit the survival of these fish in seawater cooler than their normal freezing temperatures. The type II AFP from rainbow smelt (Osmerus mordax), which is a member of the C-type lectin supe rfamily, was characterized in terms of its Ca 2+ -binding quaternary structure and the role of its single N-linked oligosaccharide. The protein core of the smelt AFP, shown through sequence homology to be a C-type lectin carbohydrate-recognition domain, was found to be protease resist ant. Smelt AFP was also shown to bind Ca 2+ , as determined by ruthenium red staining a nd a conformational change on Ca 2+ binding detected by intrinsic fluorescence. The N-linked oligosaccharide w as found to have no effect on protease resistance, dimerization, or antifreeze activity. Thus its role, if any, in the antifreeze function of this protein remains unknown. Smelt AFP was also shown to be a true intermolecular dimer composed of two separate subunits. This dimerization did not require the presence of N-linked oligosaccharide or bound Ca 2+ . Smelt AFP dimerization has implications for the effective solution concentration and measurement of its activity. This finding may also lead to new interpretation of the mechanism of ice-growth inhibition by this A FP. Keywords: antifreeze protein; C-type lectin; dimerization; glycosylation; rainbow smelt. Antifreeze proteins (AFPs) are produced by many sp ecies as an efficient means of protection from freezing. They bind directly to growing ice crystals and thus inhibit c rystal growth [1]. These proteins generate a far greater freezing point depression than would be predicted from their colligative properties. This is presumed to be due to their direct interaction with ice crystals. The AFPs only have a colligative effect on the melting point of a solution and therefore cause a thermal hysteresis. This phenomenon is the basis for quantitative measurement of AFP activity. The AFPs are structurally and evolutionarily diverse but are all functionally similar in that they bind ice and depress the freezing point. They are found in a variety of insect and plant species as well as bacteria and fungi, but fish AFPs were the first and most extensively characterized [2]. Among fish species, five structurally defined types of AFPs have been identified. The antifreeze glycoproteins found in cods and Antarctic nototheniids are composed of multiple Ala- Ala-Thr repeats, with a disaccharide linked to each Thr residue [1]. The type I AFPs found in certain flounders and sculpins are alanine-rich, amphiphilic a helices with a repeating pattern of Thr residues [3,4]. The type III AFPs have a unique globular fold with one flattened surface thought to take part in ice binding, a nd are f ound in northern and Antarctic eel pouts [5]. The recently charac- terized type IV AFP is composed of an antiparallel helix bundle homologous to an apolipoprotein, which has been only found in longhorn sculpin, Myoxocephalus o ctodec- imspinosis [6]. Type II AFPs have been shown to be homologous to C-type lectins. They are found in the serum of three teleost fishes: sea raven (Hermitripterus americanus), Atlantic herring (Clupea harengus harengus), and rainbow smelt (Osmerus mordax) [2]. They consist of a long form of the C -type c arbohydrate-recognition domain and are C-type lectin-like domains ( CTLDs) [7–9]. This domain consists of a t ightly folded hydrophobic core stabilized by three or more disulfide bonds [10]. The type II AFPs of herring and smelt appear to be very closely related but the sea raven AFP is more distinct. The smelt and herring type II AFPs exhibit near-equivalent molar antifreeze activity (thermal hysteresis), whereas the sea raven AFP appears f ar more active in generating thermal hysteresis [11]. Moreover, the smelt and herring AFPs are structurally very similar. These AFPs share 86% sequence identity in a 126-residue sequence overlap and they have very similar putative signal seq uences. They share approximately twice the percentage sequence identity with one another than either share with the sea raven protein [7]. The smelt and herring AFPs also share the Gln-Pro-Asp (galactose-binding) motif of galactose-binding C-type lec- tins. This is the centre of the carbohydrate-binding site in C-type lectins and was shown to be the ice-binding site in herring AFP [12]. Like th ese lectins, which require Ca 2+ to be bound before they can bind carbohydrate, the smelt and herring AFPs require Ca 2+ in order to bind to ice [7,13]. Smelt AFP does differ from herring AFP and other fish Correspondence to K. V. Ewart, NRC Institute for Marine Biosciences, 1411 Oxford St, Halifax, NS, B3H 3Z1, Canada. Fax: + 1 902 426 9413, Tel.: + 1 902 426 7 620, E-mail: vanya.ewart@nrc.ca Abbreviations: AFP, antifreeze protein; BS 3 , bis(sulfosuccinimdyl) suberate; CTLD, C-type lectin-like domain; Glu-C, endoproteinase Glu-C. *Present address: Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada. (Received 26 October 2001, accepted 2 January 2002) Eur. J. Biochem. 269, 1219–1226 (2002) Ó FEBS 2002 AFPs, however, in several important respects. It has an 18-residue N-terminal extension (GDTGKEAVMTGSSG KNLT) and an 11-residue C-terminal extension (VNP EVTPPSIM) not present in h erring AFP. It also has a n N-linked glycosylation site in the N-terminal extension sequence [ 13]. Therefore, the goal of this study was to investigate the structural and functional characteristics of smelt type II AFP. The effects and inter-relationship of metal ion binding, N-linked glycosylation, and quaternary structure in this protein were studied in order to b etter define its structure and function as an AFP as well as its relationship to other CTLDs. MATERIALS AND METHODS Materials N-Glycosidase F and endoproteinase Glu-C were obtained from Roche Molecular Biochemicals (Laval, Canada). Bis(sulfosuccinimdyl) suberate (BS 3 ) and Gelcode Blue (Coomassie) stain were obtained from P ierce Chemicals (Rockford, IL, USA). Sequencing-grade endoproteinase Glu-C a nd trypsin were obtained from Promega (Madison, WI, USA). Ruthen ium red was purchased from Fluka Chemicals (Ronkonkoma, NY, USA). Nitrocellulose mem- brane (0.45 lm) was purchased from Bio-Rad (Hercules, CA, USA). All other chemicals we re reagent grade. Purification of smelt AFP Blood plasma was obtained f rom a population of rainbow smelt (O. mordax) caught i n seawater a long the north- eastern coast of Newfoundland on 20 February 1997 and stored frozen until use. Approximately 2.5 mL plasma was fractionated on a 1 · 90 cm S-200 Sephacryl (Pharmacia, Uppsala Sweden) gel-filtration c olumn. Fractions contain- ing AFP, as determined by SDS/PAGE, were then applied to a 1 · 30 cm phenyl–Sepharose hydrophobic interaction column (Amersham–Pharmacia), and eluted in 20 m M Tris/ HCl, pH 8.0, buffer with a continuous descending (1–0 M ) NaCl gradient. Fractions containing AFP, as identified by SDS/PAGE, were pooled and lyophilized. The lyophilized sample was reconstituted in a minimal v olume of 0.1 M NH 4 HCO 3 buffer, pH 8.0, desalted using a PD-10 column (Amersham P harmacia), lyophilized, and stored at )20 °C. Preparation of deglycosylated smelt AFP Lyophilized N-glycosidase F was reconstituted to 1 UÆlL )1 in water. Lyophilized smelt AFP was dissolved in 25 m M Hepes, pH 7.8, to a final concentration of 4 mgÆmL )1 . Then 10 U N-glycosidase F was added to 100 lL of smelt AFP solution and incubated at 37 °C; a f urther 5 U N-glyco- sidase F added 4 h l ater, and aga in after an overnight incubation. A further 8 U was added over a period of 4 h followed by a second overnight incubation at 37 °C. The reaction was considered complete after SDS/PAGE analysis of the reaction mixture re vealed no detectable glycosylated smelt AFP using Gelcode Blue staining. A parallel control reaction was carried out by adding equivalent volumes of water t o 100 lL of the smelt AFP solution, and incubating at 37 °C for the same amount of time. Ruthenium red staining Ruthenium red dye binding was evaluated following the method of Ch aruk et al. [14] with minor modifications. Deglycosylated smelt AFP and untreated smelt AFP samples (both 4.5 lg per lane) were run on an SDS/15% polyacrylamide gel under nonreducing conditions and then electrophoretically transferred to a 0.45-lm nitrocellulose membrane. A blot section was incubated at 4 °Cfor 15 min in staining buffer (20 m M Hepes, pH 7.8, 10 mgÆmL )1 rutheniumred),followedbya15-minwash at 4 °C in wash buffer (20 m M Hepes, pH 7.8). An identical section was treated the same way except 100 m M CaCl 2 was present in both the staining and w ash buffer. Amido black staining was used to confirm protein presence and to detect bands not stained by the ruthenium red dye. Analysis of antifreeze activity Antifreeze activity was quantitated a s thermal hysteresis, which is the difference b etween the melting and freezing point of a solution. Thermal hysteresis was measured by monitoring ice crystal behavior using a nanoliter osmometer (Clifton Technical P hysics, Hartford, NY, USA). Five measurements were taken o n 130-l M solutions of deglycos- ylated and untreated smelt AFP in 25 m M Hepes, pH 7.8, using water as a blank . As a control, the thermal hysteresis of a solution of N-glycosidase F was measured in 25 m M Hepes, pH 7.8. Results were expressed a s mean ± SE. Photographs of ice crystals viewed during these measure- ments were taken at a magnification of 200·. Protease protection assays on smelt AFP Protease protection assays to compare deglyco sylated and untreated smelt AFP were performed as previously described [15] with minor modifications. Reaction volumes of 16 lL containing 4.8 lg of either untreated or deglycos- ylated smelt AFP in 10 m M Hepes, pH 7.8, and either 2 m M EDTA or 20 m M CaCl 2 were incubated for 30 min. This was followed by the addition of protease (0.1 mgÆmL )1 final concentration), or an equal volume of water, after which all samples were incubated for 3 h at room temperature. An aliquot of each reaction mixture w as resolved by SDS/ PAGE (15% gel) under reducing conditions, and stained with Gelcode Blue. In vitro chemical cross-linking Untreated and deglycosylated smelt AFP samples in HCS buffer (25 m M Hepes, pH 7.8, 150 m M NaCl, 10 m M CaCl 2 ) or HES buffer (10 m M EDTA in place of CaCl 2 ) at a final AFP concentration of 33 l M , were aliquoted into the w ells of a microtiter plate and left undisturbed for 15 min at room temperature. A serial dilution of BS 3 dissolved in either HCS or HES buffer was performed, and aliquots of each dilution were added t o corresponding AFP- containing wells. The reaction mixtures were incubated at room temperature for 1 h, then resolved by SDS/PAGE (15% gels) under reducing conditions and stained with Gelcode Blue. 1220 J. C. Achenbach and K. V. Ewart (Eur. J. Biochem. 269) Ó FEBS 2002 Determination of molecular mass by gel-filtration chromatography A TosoHaas 4-lm particle size TSK SuperSW 2000 column (4.6 · 300 mm) was equilibrated in HCS buffer and run at a flow rate of 0.3 mLÆmin )1 using an HPLC (Waters). The column was calibrated using protein molecular-mass stand- ards. Approximately 15 lg untreated smelt AFP was applied to the column in duplicate runs. Similar samples of deglycosylated AFP were applied as well. All proteins were detected by monitoring A 230 . Fluorescence measurements Intrinsic fluorescence was measured using an Amin co Bowman series 2 spectrofluorimeter at room temperature. Untreated and deglycosylated s melt AFP samples were diluted to equimolar concentrations in 25 m M Hepes, pH 7.8, containing 1 m M EDTA. Emission spectra were recorded with a 2-nm/s scan rate in duplic ate for each trial with an excitation wavelength of 280 nm (4 nm bandpass). CaCl 2 was added by pipetting a 0.5- M solution directly into the sample cuvette with thorough mixing followed by a 10 min incubation at room temperature. A 0.5- M solution of EDTA (pH 8.0) was added in a similar fashion to test the reversibility of any Ca 2+ effect. Spectra were corrected for the resultant dilutions. RESULTS Deglycosylation of smelt AFP To study the effect of the N-linked o ligosaccharide of smelt AFP, it was removed from the protein enzymatically using N-glycosidase F. The reaction evaluated using SDS/PAGE was f ound to result in a band of reduced size (17 kDa) compared with the untreated band (22 kDa) (results not shown). The 17-kDa molecular mass corresponds to the 17.4-kDa calculated mass o f the herring AFP sequence (from cDNA) with the predicted signal sequence removed. This result implies that the smelt AFP has no prosequence and co nsists of the complete sequence p redicted from cDNA minus the signal. Ruthenium red staining To detect Ca 2+ binding by smelt AFP, both untreated and deglycosylated smelt AFP were blotted as discussed in Materials and methods and stained with a 10 mgÆmL )1 solution of ruthenium red. This dye was shown to bind both the untreated and deglycosylated smelt AFP (Fig. 1) as well as the known Ca 2+ -binding molecular mass standard b-lactoglobulin (not shown). The specificity of binding is demonstrated by the absence of detectable ruthenium red staining when CaCl 2 is added to the staining and washing buffer (Fig. 1). Measurement of antifreeze activity Antifreeze ac tivity was measured on equimolar amounts of deglycosylated and untreated smelt AFP to determine whether the N-linked oligosaccharide had any effect on this activity. A faceted ice-crystal morphology signifies the presence of antifreeze activity. The rounded ice crystal formed in the control sample c ontaining only N-glycosi- dase F shows that the enzyme does not display antifreeze activity (Fig. 2). Equimolar solutions of deglycosylated and untreated smelt AFP displayed similar faceted ice crystal morphology (Fig. 2). The antifreeze activity of the untreat- ed and d eglycosylated samples, quantitate d as thermal hysteresis, were 0.027 ± 0.003 and 0.026 ± 0.003, respect- ively. These are not significantly different (P < 0.001). As expected, the enzyme control sample showed no activity, with a hysteresis value of )0.001 ± 0.002. Protease protection assays To determine whether Ca 2+ or the N-linked oligosaccha- ride has any effect on the protease susceptibility of smelt AFP, both untreated and deglycosylated smelt AFP were digested with Glu-C and trypsin in the presence and a bsence of Ca 2+ . Both enzymes generated some proteolysis near the N-terminus and/or C-terminus of the protein leaving a large central AFP fragment intact. The sizes o f the digestion fragments were all greater than 14 kDa, w hich is the approximate size of the core CTLD. The addition of Ca 2+ at a concentration of 20 m M had no effect on the p roteolysis seen with either deglycosylated o r untreated smelt AFP (Fig. 3 A,B). This suggests that the CTLD of smelt AFP is protease resistant both in the presence and absence of Ca 2+ and with a nd without the N-linked oligosaccharide. Similar digestion patterns were also seen in both the untreated and deglycosylated smelt AFP trials using either protease. Digestion of either deglycosylated or un treated smelt AFP formed products that were 1 kDa and 2 kDa smaller than undigested smelt AFP. The size differences between the digestion fragments of untreated and deglycosylated AFP Fig. 1. Ruthenium red staining of smelt AFP. Ruthenium red (RR) staining of deglycosylated (D) smelt AFP and untreated (U) smelt AFP was carried out as described in Materials and methods. Blots shown are stained with a mido black (Amido), ruthenium red (RR) or ruthenium red in the p resence of 100 m M CaCl 2 (RR + Ca 2+ ). Molecular-mass marker sizes (kDa) are indicated. Ó FEBS 2002 Characterization of smelt antifreeze protein (Eur. J. Biochem. 269) 1221 are all 5 kDa, which corresponds to the apparent size of the carbohydrate determined in a separate experiment (Fig. 1). Taken together, these results indicate that the proteases digest both untreated and deglycosylated forms of smelt AFP in close, if not ide ntical, locations. These results also indicate that protease digestion under these conditions does not cleave between the Asn residue carrying the N-linked oligosaccharide and the core CTLD. Bands corresponding to Glu-C and trypsin enzymes were identified on the basis of molecular mass. The bands smaller than 14 kDa in the trypsin digests were shown to be trypsin autolysis products in a separate control experiment containing trypsin alone (not shown). Intrinsic fluorescence Modulation o f protein conformation on addition of Ca 2+ was monitored by intrinsic fluorescence. Excitation at 280 nm produced an emi ssion spectrum with a k max of 343 n m from % 30 l M samples of both untreated (Fig. 4A) Fig. 2. Analysis of antifreeze a ctivity. Antifreeze activity was evaluated qualitatively by monitoring ice crystal morphology in solutions con- taining deglycosylated and untreated smelt AFP (both 130 l M )as described i n Materials and metho ds. The top row of p hotos is of ice crystals with their c axis in the plane of the page. The bottom row shows ice crystals with their c axis normal to t he page. Fig. 3. Proteolytic digestions of deglycosylated and untreated smelt AFP in presence and absence of Ca 2+ . Both deglycosylated and untreated smelt AFP were incubated with protease in the presence or absence of 20 m M Ca 2+ as described in Materials and methods. All reaction products were analyzed by SDS/PAGE and stained with Gelcode Blue. (A) AF P digested with Glu -C. Bands corresp onding to Glu-C , untreated ( UT) AFP (UT AFP), and deglycosylated AFP (DG AFP) are identified. Proteolytic fragments are seen as lower-molecu lar-mass bands in Glu-C-contain ing lanes. ( B) AFP digested with trypsin. Bands correspond ing to trypsin, untreated AFP (UT AFP), and deglycosylated AFP (DG) are id entified. Bands smaller than 1 4 kDa in lan es containing trypsin were shown to b e trypsin autolysis products (trypsin-only digest not shown). Molecular-mass marker sizes (kDa) are indicated. Fig. 4. Effect of Ca 2+ on the intrinsic fluorescence of deglycosylated and untreated smelt AFP. (A) Emission spectra of intact (u ntreated) AFP (UT AFP). (B) Emission spectra of deglycosylated AFP (DG AF P). Spectra were recorded for solutions of 30 l M smelt AFP in 25 m M Hepes, pH 7.8, containing 1 m M EDTA, before and after the ad dition of CaCl 2 (10 m M ). Each spectrum shown is the average of two suc- cessive recordings. All measurements were taken with excitation at 280 n m and a 2-nmÆs )1 scan rate at ambient temperature. 1222 J. C. Achenbach and K. V. Ewart (Eur. J. Biochem. 269) Ó FEBS 2002 and deglycosylated (Fig. 4B) smelt AFP. The emission intensity was shown to increase significantly i n both samples on addition of Ca 2+ to a final concentration of 10 m M .The effect was fully reversible in both samples, with the addition of EDTA to a final c oncentration of 18 m M (not shown). No blue or red shift was observed in the emission spectra of either sample on addition of Ca 2+ or EDTA. Detection of dimerization To determine the quaternary structure of smelt AFP in solution, AFP (33 l M ) was incubated in the presence of a range of concentrations of BS 3 , a water-soluble homo- bifunctional cross-linking agent, in the presence of 10 m M CaCl 2 .IntheabsenceofBS 3 , a 22-kDa band corresponding to the smelt AFP monomer was observed (Fig. 5A). A BS 3 concentration of 0.16 m M was sufficient to gen erate the accumulation o f a larger protein form. W ith increasing concentrations of BS 3 , the disappearance of the monomer band corresponded to the gradual increase in intensity of a higher-molecular-mass band. The apparent molecular mass of this band was 38 kDa, which is substantially higher than the molecular mass of the monomer band, indicating the presence of a dimer. The expected mass of a smelt AFP dimer is 44 kDa but, on SDS/PAGE, the covalently cross- linked dimer would be expected to migrate faster than its actual mass would predict because o f limited linearization in SDS. The value of 38 kDa is consistent with such a d imer band. A small amount of a high-molecular-mass aggre gate was evident at the top of the SDS/PAGE gel lane co ntaining the sample with t he highest concentration (20 m M )ofBS 3 . This appears to be an artefact of high cross-linker concen- tration a nd was not taken as evidence of any larger protein aggregate. To determine whether the oligosaccharide plays a role in dimerization, the experiment was repeated on deglycosylated smelt AFP (Fig. 5B). Dimerization was evident with monomer bands of 18 kDa and dimer bands of 33 kDa. To examine whether Ca 2+ binding was required for dimerization, smelt AFP was incubated with BS 3 in buffer containing 10 m M EDTA instead of CaCl 2 .Inthe absence of added Ca 2+ , dimerization was again observed (Fig. 5C). The molecular mass of smelt AFP determined by HPLC gel-filtration analysis was 50 kDa (Fig. 6 ). As the molecular mass of the protein on SDS/PAGE is 22 kDa, the value obtained b y g el filtration is consistent with a dimer. There was no e vid ence of l arge r aggregates, a s determined by absorbance at 230 nm, nor were there any detectable peaks corresponding to the monomer size (22 kDa). These results are in agreement with the cross-linking experiments indica- ting that the native smelt AFP is fully dimerized. Deglycos- ylated AFP was also analyzed on the same column and found to have a molecular mass of 41 kDa, which also indicates dimer formation (not shown). Fig. 5. Chemical cross-linking of smelt AFP. Smelt AFP (33 l M ) was cross-linked using BS 3 as described in Materials and methods. After the 1 h incubation, reaction c ontents were analyzed using SDS/PAGE under reducing conditions and stained using Gelcode Blue. Lanes 1–9 in all gels correspond to BS 3 concentrations of 0, 0.16, 0.3, 0.6, 1.25, 2.5, 5, 10, and 20 m M , respectively. (A) I ntact AFP in the presence of CaCl 2 .(B) Deglycosylated AFP in the presence of CaCl 2 . (C) Intact AFP in t he presence of EDTA. P osit ions of dimers (D) and monomers (M) and the molecular-mass marker sizes (kDa) are indicated. Fig. 6. HPLC gel-filtration analysis of smelt AFP. Smelt AFP was applied to t he colum n un der t he co nditio ns described in Materials and methods. The elution times of molecular-mass marker proteins (kDa) are indicated by arrows. Ó FEBS 2002 Characterization of smelt antifreeze protein (Eur. J. Biochem. 269) 1223 DISCUSSION Intermolecular dimer formation by the smelt AFP makes it unique among the fish AFPs. Other AFP types, s uch as type I, IV, and the antifreeze glycoproteins, appear to exist as monomers. Similarly, type III AFP is monomeric, and shown not to self-associate when binding ice [16]. A type III AFP f rom an Antarctic eel p out ( Rhigophila dear borni) contains a tandem repeat of the domain that comprises other type III AFPs on a single chain [17]. However, the type III AFP, referred to as a n intramolecular dimer, is i n effect a monomer (single polypeptide chain) and not a true intermolecular dimer. The AFPs of winter rye (Secale cereale) w ere found to form larger intermolecular com- plexes, a lthough the precise subunit composition in the hetero-oligomers containing different A FPs from t his plant is unknown [18]. The dimerization of smelt AFP is intriguing because the homologous sea raven type II AFP was found to exist as a monomer in solution [19]. Dimerization of smelt AFP is unlikely to increase activity by increased ice binding because only a dimer with perfectly aligned ice-binding sites would have enhanced binding to ice and this is not likely to be the case. This may be relevant to the calculation of smelt AFP activity. The activity of smelt AFP is about one-third of that of sea raven AFP on a monomer concentration basis [11]. As sea raven and herring AFPs bind to ice at a distinct site on the CTLD [12,21], it is possible th at t he activity difference simply r eflects the difference in ice binding. However, if the dimerization of smelt AFP e ffectively prevents one subunit from binding ice, this could a ccount f or some or all of t he difference in activity calculated on a monomer basis. The gel-filtration and cross- linking results shown here suggest that smelt AFP is fully dimerized under physiological conditions, with very low or undetectable levels of monomeric protein. Because smelt AFP is fully dimerized, it would be more appropriate to calculate its activity on the basis of dimer molarity t han on a monome r basis . Although the dimerization site of smelt AFP cannot be determined from th e results of this study, it is unlikely that the dimerization is due to intermolecular disulfide bonds. Smelt AFP migrates on SDS/PAGE under nonreducing conditions with a monomeric size, as shown in the ruthenium red-binding assay (Fig. 3). In addition, all of the cysteine r esidues are conserved among the three type II AFPs and w ere shown to f orm intramolecular disulfide bonds in the monomeric sea raven AFP structure [8]. Because it was possible to chemically cross-link the smelt AFP dimers using the homobifunctional cross-linking agent BS 3 , dimerization must result in the p lacement of two primary amines, from either lysine side chains, or the N-terminus, a maximum distance of 11.4 A ˚ apart. This value corresponds to the spacer arm length of the BS 3 . Several of the soluble C-type lectins associate to form dimers [22–25]. Therefore, t he dimerization of smelt AFP is consistent with other members of the C-type lectin super- family and is well in keeping with the soluble lectins among them. By analogy with other C-type lectins known to dimerize, t here could be several types of dimerization surfaces [22,23]. Dimerization or multimerization are com- mon characteristics of C-type lectins, especially those involved in the a cute-phase response o f host i mmune defense such as the collectins. In such cases, multimerization serves to enhance the avidity and pattern recognition of such lectins towards pathogen carbohydrate structures [26]. The inorganic dye ruthenium red has been shown t o selectively bind to Ca 2+ -binding sites of several proteins, including the herring AFP [12–15]. Like the herring A FP, smeltAFPwasshowntobindtheCa 2+ analogue, ruthenium red, suggesting the presence of a Ca 2+ -binding site. Further evidence for direct Ca 2+ binding by smelt AFP was obtained using intrinsic fluorescence. An increase in intrinsic fluorescence emission intensity was shown to result from Ca 2+ addition, consistent with the result of similar experiments on herring AFP [15]. This increase indicates a change in the environment of tryptophan residues in the protein on Ca 2+ addition, which is normally indicative of a conformational change. The number of bound Ca 2+ ions per smelt AFP monomer remains unknown. However, because smelt AFP is identical with that of herring in the positions corresponding to lectin Ca 2+ -binding sites [7], it is reasonable to suggest that the smelt AFP binds a single Ca 2+ in the same way as the herring AFP [15]. In comparison w ith o ther C-type lectins and he rring AFP, smelt AFP appears to be a remarkably p rotease- resistant protein in both the presence and absence of Ca 2+ ions. Digestion with trypsin or endoproteinase Glu-C resulted in the formation of two slightly smaller digestion fragments regardless of whether or no t C a 2+ was p re sent. The large size of the digestion fragments indicates that most of the smelt AFP is unavailable for protease digestion under the conditions tested. The sizes of the digestion fragments were on average only % 1–2 kDa smaller than the undi- gested smelt AFP, suggesting that cleavage of only t he extended N-terminus and C-terminus occurred, leaving the remaining CTLD. These results are consistent with the hypothesis that smelt AFP monomer is composed primarily of a single CTLD. However, in similar studies, the CTLD- containing herring AFP and chicken hepatic lectin did n ot show the same level of protease resistance in the absence of bound Ca 2+ [15,27]. The dimerization shown to o ccur between smelt AFP monomers may enhance protease resistance by shielding residues in the dimerization interface. A conformational change s hown to occur in herring AFP on Ca 2+ binding generated a shift from p rotease sensitivity to protease resistance on Ca 2+ binding [15]. However, in t he present investigation of smelt AFP, there was evidence for a protease-resistant CTLD core in the absence Ca 2+ . The addition of Ca 2+ afforded no further protection from proteolysis. It is clear from the fl uorescence expe riments t hat apo-(smelt AFP) undergoes a conformational change i n response to Ca 2+ binding, which in other lectins and herring AFP is necessary for protease resistance. It is highly unlikely that sites susceptible to proteolysis in C a 2+ -free herring AFP and related C-type lectins would be less so in smelt AFP as a result of any residue differences because equiv- alent protease resistance and fragment patterns were e vident when trypsin was used instead of Glu-C. The cross-linking experiments revealed that smelt AFP d imerizes in the presence or absence of EDTA, indicating that the protein remains dimerized in the absence of bound Ca 2+ ions. This may protect susceptible surfaces from protease digestion in smelt AFP, with or without Ca 2+ . An interesting difference between smelt AFP a nd the other known AFPs of fish, including the other type II AFPs, is the presence of an N-linked oligosaccharide located on 1224 J. C. Achenbach and K. V. Ewart (Eur. J. Biochem. 269) Ó FEBS 2002 Asn18 [19]. The biological roles of the N-linked oligosac- charides from many proteins have been studied (reviewed in [28,29]). N-linked oligosaccharides have been shown to enhance the thermal stability o f proteins, modulate and stabilize protein secondary structures such as b turns, mediate interc ellular t ransport of polypeptides, modulate protein half-life, and facilitate protein–protein interactions [28–31]. To examine possible roles of the N-linked oligo- saccharide o f smelt AFP in native protein structure and function, deglycosylated and untreated smelt AFP were compared in t erms of dimerization, Ca 2+ binding, protease resistance, and antifreeze activity. Enzymatic cleavage with N-glycosidase F resulted in an apparent reduction in molecular mass of % 5 kDa. The removal of the carbohy- drate moiety had no effect on any of the characteristics of smelt AFP that were investigated. However, the possibility of alternative and untested roles of the N -linked oligosac- charide such a s enhanced protein half-life or recognition by endogenous lectins in vivo cannot be ruled out. Rainbow smelt are unique among bony fishes in that they have been shown to p roduce large amounts o f glycerol (upto0.4 M ) in response to subzero temperatures. At these concentrations, glycerol generates a substantial colligative freezing point depression and depresses the body freezing temperature of the smelt along with the noncolligative activity of the smelt AFP [32]. Although smelt AFP clearly contributes to the depression of the serum freezing tem- perature, it does not appear to be the major contributor to this effect. It would therefore be interesting to determine whether the AFP has a separate undiscovered activity in smelt plasma, in which the N-linked oligosaccharide and dimeric character play more central roles. Smelt AFP has been characterized in terms of Ca 2+ binding, function of the N-linked o ligosaccharide, and quaternary structure. This study demonstrates that Ca 2+ imparts a con formational change w hen bound to smelt AFP in the same w ay as for h erring AFP. However, unlike herring AFP, the core CTLD of smelt AFP is protease resistant even without Ca 2+ bound. Smelt AFP is unusual among general AFPs in that it is N-glycosylated and dimeric. The smelt type II AFP does not bind common carbohydrates as many lectins do [7], but its similarity to the soluble C-type lec tins is evident in its structural character- istics and function. The mechanistic implications o f the observed dimerization on smelt AFP ice binding requires further study. ACKNOWLEDGEMENTS We thank Devanand Pinto (NRC IMB) for helpful review of the manuscript. We also thank a number of IMB colleagues for their kind assistance, including Robert Richards for helpful d iscussion, Shawna MacKinnon for use of her HPLC, Denise LeBlanc for loan of a size exclusion HPLC column, Steve Locke for trypsin, and Neil Ross for time on his fluorimeter and patient instruction in software use for the instrument. We are grateful to A/F Protein Canada Inc. for their generous donation of smelt blood plasma. This research was supported by NRC IMB. This is NRC p ublication number 42345. REFERENCES 1. Yeh, Y. & Feeney, R.E. (1996) Antifreeze proteins: structures and mechanisms of function. Chem. Rev. 96, 601–617. 2. Ewart, K.V., Lin, Q. & Hew, C.L. (1999) Structure, function and evolution of antifreeze proteins. Cell. Mol. Life Sci. 55, 271–283. 3. Hew, C.L., Joshi, S., Wang, N.C., Kao, M.H. & Anantha- narayanan, V.S. 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