416
Biochimica et Biophysica Acta~ 1076(1991) 416-420 © 1991 Elsevier Science Publishers B.V. 0167..4838/91/$03.50 ADONIS 0167483891001I4A
BBAPRO 33844
Enhancement of insect antifreeze protein activity by antibodies D i n g W e n W u , J o h n G , D u m a n a n d Lei X u Department of Biological Sciences, University of Notre Dame, Notre Dame, IN (U.S.A.) (Received 13 July 1990) (Revised manuscript received 12 October 1990)
Key words: Antifreezeprotein;Coldtolerance;Insectantifreeze;Thermalhysteresisprotein Antifreeze proteins, produced by many cold water marine teicost fish and terrestrial arthropo~ (insects, spiders, etc.), inhibit ice crystal growth by a non-ealligative mechanism, probably by adsorbing onto the surface of potential seed ice crystals and thereby blocking growlh at preferred growth sites, in this study it is demonstrated that the activity of two insect antifreeze proteins is greatly increased by the addition of specMic rabbit polydonal antibodies to the antifreezes. A model is presented which suggests that the enhancement occurs because the antifrceze-antibody complex, being much larger than the antifreeze protein alone (a minimal 7-8-fold increase in size), blocks a larger area of the ice crystal surface and extends fmltber above the. sm'face, thus requiring the temperahn-e to be further lowered before e~stal grog~h proceeds. This idea is further supported by the fmding that addition of goat anti-rabbit IgG to the antifreeze protein + anti-antlfreeze protein antibody complexes further enhanced activity. lntrodnefian Antifreeze proteins have been best studied in cold water marine teleost fishes. (for a review see Refs. i-3). These antifreezes inhibit ice crystal growth by a noncolligative mechanism whereby the freezing point is depressed below the melting point, thus producing a so-called thermal hysteresis. Evidence indicates that the proteins exert their antifreeze effect by an adsorptioninhibition mechanism in which they adsorb onto the surface of potential seed ice crystals, the-eby inhibiting crystal growth [5-7]. Purified antifreeze proteins demonstrate a hyperbofic relationship between protein concentration and thermal hysteresis activity, with the plateau of activity for the more active forms of fish antifreeze proteins at approx. 1.5 o C. Thus higher levels of the antifreeze provide sufficient freezing point depression, in combination with the colligative depression resulting primarily from inorganic ions, to lower the freezing point of the body fluids of the fish below that of seawater. Antifreeze proteins are also known to be produced by terrestrial arthropods, including many insects [8]. spiders [9], a centipede and an Antarctic mite [10] and they have been purified from four species of insects [11-16]. These include the antifreeze proteins used in Correspondence:J. D,,man; Dcpamnentof BiologicalSciences,Universityof Notre Dame.Notre Dame,1N 46556,U.S.A.
this study, from the larvae of the beetles Tenebrio molitor and Dendroides canadensis. The relationship between activity and concentJation for Tenebrio antifreeze proteins is similar to that of the fish [17,18]. Purified Dendroides antifreeze proteins are more active, with
maximal hysteresis activity of purified protein at -~ 2.5°C [8], however mid-winter mean hemolymph hysteresis values of Dendroides populations are typically 3 to 6 ° C with some individuals having 8 - 9 ° C [19]. Recent work OVu and Duman~ unpublished data) has shown that certain proteins can activate the purified Dendroides antifreeze proteins to produce levels of hysteresis comparable to those seen in the winter hemolymph. In the study presented here we show that specific antibodies to insect antifreeze proteins also increase their hysteresis activity. Materials and Methods Preparation of antiserum. Larval Dendroides canadensis were collected from partially decompo,'ed logs in
woodlots near South Bend, IN. Antifreeze proteins were purified OVu, Duman, Cheng and Castellino, unpublished data), and a fraction containing a mixture of two antifreeze proteins (H1 and H2) were used to raise antiserum in male New Zealand white rabbits [20]. (Note that Dendroides hemolymph contains four similar/mmunological!y indis'dnct antifreeze proteins.) Tenebrio molitor, originally purchased from Connecticut Valley Biological Supply, were cultured in the
417 laboratory on a diet of wheat bran. Larger larvae were periodically acclimated at 10*C and a photoperiod of 8 light/16 dark to induce antifreeze protein production. Antifreeze proteins were purified [18] and used to raise antiserum in male New Zealand white rabbits (Xu, Duman and Goodman, unpublished data). ELISAs were used to determine the antibody titres of the two antisera [21]. Peroxidase conjugated goat antirabbit IgG antiserum (Calbiochem) was used as the second antibody for color development. The final detectable concentration of anti-Dendroides antifreeze protein antiserum was a 2TM dilution, and that of the antiTenebrio antifreeze protein antiserum was a 215 dilution. Preparation of IgG and Fab fragments. Total IgG was prepared from the rabbit Tenebrio antifreeze antiserum using an AFFINICA protein A / G column (Schleicher and Schuell) with a binding buffer of 1.5 M glycine, 3 M NaC (pH 8.9) and an elution buffer of 0.2 M glycine-HCl (pH 2.5) at a flow rate of 0.5 ml/min. Fab fragments [22] of the IgG were prepared by digesting the IgG with papain for 10 h (10/tg papain/mg IgG) at 37°C in a buffer of 100 mM sodium acetate, 50 mM cysteine and 1 mM EDTA (pH 5.5). Digestion was terminated by adding 75 mM iodoacetamide. Thc preparation was dialyzed against the binding buffer as used for IgG preparation and the Fab fragments were purified using an AFFINICA protein A / G column as described ::hove. Both the IgG and Fab fragments ran as single bands on SDS-PAGE (non-reducing conditions), indicating their purity. An ELISA (as described above) was used to determine the titre of the purified IgG. Beginning with an IgG concentration of 10 mg/ml, a 2-fold dilution series was run. The end point was 217. Effect of antibodies on antifreeze protein activity. Thermal hysteresis antifreeze activity was determined using the freezing point-melting point difference (thermal hysteresis) technique [23]. To determine the effect of the antibodies on antifreeze protein activity, the thermal hysteresis of the antifreeze proteins was determined at various concentrations, with and without the presence of antisera. Antisera was added to a final concentration of 0.5~ (v/v) in these experiments. Preimmunization rabbit sera was used as a control. To determine the effect of variations in antibody concentration on antifreeze protein activity, Dendroides antifreeze at a final concentration of 2.5 m g / m l was treated with various concentrations of antiserum, ranging in fmal concentration from 0 to 200%. Antiserum concentrations up to 50% were achieved by diluting the antiserum with buffer as appropriate, and then mixing the diluted antiserum 1/1 (v/v) with a 5 m g / m l antifreeze protein solution. For example, to get a final concentration 25% antiserum and 2.5 m g / m l antifreeze, the original antiserum was diluted 1/1 with buffer, then 1 vol. of the diluted antiserum was added to 1 vol. of a
5 m g / m l antifreeze solution. Concentrations of antiserum above 50% were achieved by freeze drying a volume of the antiserum, redissolving the antiserum in a smaller volume of buffer, and then adding this to the antifreeze protein solution as appropriate. For example, to get a 200% final antiserum solution 1 vol. of antiserum was freeze dried, redissolved in 1 / 4 vol. of buffer, and this mixed 1/1 with a 5.0 mg/ml soltion of antifreeze protein. Effects of P A N S O R B I N and goat-anti rabbit IgG on the activity of anti-antifreeze antiserum treated antifreeze protein. PANSORBIN (Staphylococcus aureus cells coated with protein-A, purchased from Calbiochem. Binding capacity: 2.05 rag human l g G / m l of cell suspension.) at a final concentration of 33% was added to an aqueous solution containing Dendroides hemolymph (33%, v/v, final concentration) and anti-Dendroides antifreeze-antiserum (33%, v/v, final concentration). The mixture was incubated at room temperature for 30 rain, centrifuged at 7 0 0 0 × g for 5 rain at 0 ° C and the supernatant removed. Thermal hysteresis activity of the superuatant was determined. Solutions of Dendroides hemolymph diluted to 33% final concentration with Tris-NaCl buffer, and of hemolymph (33%) plus antiserum (33%) were used as controls. The effect of addition of goat-anti rabbit lgG (Calbiochem) on the activity of a mixture of anti-Dendroides antifreeze antiserum plus Dendroides antifreeze protein was determined. Goat-antirabbit antiserum (final concentration of 33%) was added to anti-Dendroides antifreeze antiserum (final concentration of 33%) and Dendroides hemolymph (final concentration of 33%). The mixture was incubated at room temperature for 30 rain and the activity determined. Controls were diluted Dendroides hemolymph (33%) and Dendroides hemolymph (33%) plus antiserum (33%). Effects of anti-Tenebrio antifreeze-lgG and Fab fragments on Tenebrio antifreeze protein activity. Purified Tenebrio antifreeze (final concentration of 10 mg/ml) was treated with either lgG (final concentration: 5 mg/ml) prepared from anti-Tenebrio antifreeze antiserum (as described above) or purified Fab fragments (final concentration: 3 mg/ml) from the lgG and the thermal hysteresis activity was determined. The control was Tenebrio antifreeze protein (10 mg/ml) alone. Statistics. One-way analysis of variance (ANOVA), and in some eases multiple comparisons tests frukey's) were used to analyze the data for statistical significance. R ~
Enhancement of AFP activl.O, by an:ibodies. Contrary to our expectations, when anti-Dendroides antifreeze antiserum was added to the aqueous solution of Dendroides antifreeze protein, the thermal hysteresis activity increased (Fig. 1). Note the approximate doubling of
418 4-
¢J
3.0 •
2.o.
i
-r =
J
2.
J= on
1.0'
J 0.0'
1'0
2'0
0.0
3'0
Fig. l. Enhancing effect of addition of anti-Dendroides antifreeze antiserum (final volume of 0.5~, v/v) on the thermal hysteresis antifreeze activity of Dendroides antifreeze protein. ~ activity at various concentration~ of antifreeze protein in distilled water; ×, activity at various concentrations of antifreeze protein with addition of 0.5~ prcimmouized rabbit serum; and A, activity at various concentrations of antifree=e protein with addition of 0.5~ anti.Dendroides antifreeze protein antiserum. Addition of antiserum caused a statistically significant increase in activity at all concentrations tested (P < 0.05; one-wayANOVA).
activity with addition of antibody at antifreeze concentrations above - 3 m g / m l and the presence of activity at very low antifreeze protein concentrations where without antibody addition there was none. The slight increase in activity resulting from addition of preimmunization rabbit serum is probably due to the addition of salts in the serum to the antifreeze protein in distilled H zO, an effect we have previously recognized. (Note that the preimmunization serum does not have hysteresis activity.) To determine whether the antigen-antibody complex was soluble, Dendroides hemolymph (containing antifreeze protein) was added to an equal voL of anti-Dendroides antifreeze antiserum and the mixture was centrifuged at 6 0 0 0 × 8 ( 0 ° C ) for 15 min. Precipitate was not visible and thermal hysteresis activity was not lost. An even more exaggerated effect was observed with addition of anti-Tenebrio antifreeze antiserum to Tenebrio antifreeze protein (Fig. 2). The hysteresis activity at an antifreeze protein concentration of 2 m g / m l increased nearly 5-fotd. T h e effect on thermal hysteresis of addition of different ratios of antibody to antifreeze protein was investigated by varying the percentage of anti-Dendroides antifreeze antiserum added to a 5 m g / m l concentration of Dendroides antifreeze proteht (Fig. 3). An antiserum concentration of 0.02~% had no effect on activity, howe:'er, 0.05 and 0.50% antiserum caused a statistically significant increase (Tukey's). Interestingly, higher antiserum levels caused a significant decrease in activity as compared to that at 0.50%, and did not result in a significant, increase in activity above that without antiserum. Consequently, only lower ratios of a n t i b o d y / antifreeze produced the enhancement. Although the ex-
210
1.0
AFP (mg/ml)
AFP (mg/ml)
Fig. 2. Enhancing effect of addition of anti-Tenebrio antifreeze antiserum (final volume of 0.5~. v/v) on the thermal hysteresisantifreeze activity of Tenebrio antifreeze proteins. [3, activity at various concentrations of Tenebrio antifreeze protein either in distilled water or with addition of 0.5~ (v/v of final volume) prcimmanized rabbit serum; and a, activity at various concentrations of antifree-..eprotein with addition of 0.5~ (v/v of final volume) anti-Tenebrio antifreeze protein antiserum. Addition of antiserum caused a statistically significant increase in activity at all concentrationstested (P < 0.05; one-way ANOVA).
planation for the lack of enhancement at higher antibody/antifreeze ratios is unknown to us, one possibility is that at higher ratios more than one antibody molecule may bind each antifreeze molecule and thus produce a steric hindrance which precludes adsorption of the antifreeze protein to ice. Effects of PANSORBIN, anti-lgG and Fab fragments on activity. As shown in Table I, addition of PANSORBIN to a solution containing Dendroides antifreeze protein (hemolymph) and antifreeze antiserum eliminated the hysteresis activity. The protein-A coating the bacterial cells should, and apparently did, bind the antifreeze protein antibody complexes. The cells were then centrifuged out, titus removing the activity. (Notc, activity disappeared upon addition of PANSORBIN, even prior to centrifugation.)
°i 3: t-
O 0.0
0.25
0.5
50
Concentration
100
150
200
of A S (%)
Fig. 3. Effect of addition of various concentratioqs (~, v/v, of final volume) of anfi-Dendroides antifreeze protein antiserum on the thermal hysteresisantifreeze activity of Dendroidesantifreeze protein (2.5 mg/ml, final concentration). Concentrations of antiserum below 0.55 were most effective.
419 TABLE I Effects of PANSORBIN, anti-lg6 and Fab fragments on activity
(A) Effect of addition of PANSORBIN or goat anti-rabbit lgG antibodies on the thermal hysteresis antifreeze activity of diluted (33%) Dendroides hemolymphtreated with anti-Dendroides antifreeze antiserum. (B) Effects of addition of IgG from anti-Tenebrio antifreeze antiserum, or Fah fragments from this IgG, to Tenebrio antifreeze protein. Values reported are means:i:S.D. Values were compared using one-wayANOVA.WheneverANOVA proved significant, further analysis was conducted using Tukey's multiple comparisons test. Samplenmans having the same Tukey's rank are not significantly different from one another, but are significantlydifferent from sample means with different Tukey's ranks. Sample (A) 33% Dendroides hemolymph 33% hemolymph+33% antiserum 33% hemolymph+ 33% antiserum + PANSORBIN 33% hemolymph+ 33% antiserum + anti-lgG antiserum (B) Tenebrio antifreeze protein (10 mg/ral) Tenebrio antifreeze protein + lgG (5 mg/rul) Tenebrio antifreeze protein + Fab fragments(3 mg/ml)
growth front proceeds as a circular plate as shown in Fig. 4a, Antifreeze proteins inhibit this preferred growth by adsorbing onto the ice surface, thereby increasing the radius of curvature of the growth fronts (Fig. 4b), raising the interracial free energy and requiring a greater amount of undercoofing to achieve significant growth. Based on this adsorption-inhibition m e c h a n i s m of antifreeze protein activity [6,7], it is likely that the increased activity provided by the addition of antibody to produce soluble antifreeze-antibody complexes is due to
TH ( o C)
Tukey's rank 1.20:i:0.09 C 1.72+0.08 B
0.00+0.00
D
2.86+0.10
A
1.705:0.20
b
2.725:0.51
a
1.71 + 0.70
b
Addition of goat anti-rabbit lgG antiserum to the solution of antifreeze protein and anti-antifreeze antiserum (containing the antifreeze protein + antiantifreeze protein I g G complex) further increased activity (Table I), presumably due to formation of the antifreeze protein + anti-antifreeze protein lgG + anti-lgG complex. As expected, addition of total IgG from anti-Tenebrio antifreeze antiserum to Tenebrio antifreeze protein increased activity (Table I). However, addition of purified Fab fragments from this same lgG did not affect the activity. (The Fab fragments of the IgG contain the antigen binding sites.)
Discussion The addition of specific polyclonal antibodies enhanced the thermal hysteresis antifreeze activity of the Dendroides and Tenebrio antifreeze proteins at all antifreeze concentrations tested. As expected the addition of PANSORBIN to the antifreeze-antibody solutions removed all activity, demonstrating that the antifreezeantibody complexes were present. In contrast, addition of goat anti-rabbit I g G antiserum to solutions containing antifreeze-antibody complexes resulted in a further increase in activity. A model which attempts to explain the results is shown in Fig. 4. Normally ice crystals preferentially grow perpendicular to their C axis by addition of water molecules, probably to step sites. The
surface o f an i c e c r y s t a l
m
growth of the ice crystal antifreezeprotein
d~N
anti-AFP IgG
~II~,
anti-lgG antibody
Fig. 4. Diagrammatic representation of the adsorption - inhibition model of antifreeze protein activity with an elaboration thereon m explain the antibody enhancement effect. (a) Normal growth of ice (antifreeze protein not present) at a step site with the new growth front forming as a circular plate at a temperature just below the melting point. (b) Inhibition of growth by adsorbed antifreeze proteins. This increases the radius of curvature of the growth fronts and requires a decrease in temperature for significant growth to occur. Antifreeze proteins may eventually be overgrown,perhaps leading to rapid growth unless inhibited by further adsorption of antifreeze proteins. (c) Additional inhibition of crystal growth is provided by adsorption of antifreeze protein to which anti-antifrecee protein 18G is attached. At the low ratios of antibody/antifrceze protein used in these studies most antibody should hind two antifreeze proteins, as shown here (d) Even greater inhibition of growth results from adsorption of antifreeze protein plus anti-antifreezeprotein |gG plus anti-lgG antibody complexes.
420 enhanced blocking of potential growth sites by these complexes (Fig. 4c). The antibody, mainly IgG, is much larger (--- 150 kDa) than the antifreeze proteins (15-22 kDa), and one IgG can bind a maximum of two antifreeze proteins. (At the low antibody/antifreeze ratios generally used in this study most of the antibody molecules are expected to bind two antifreeze protein molecules.) Thus, if the antibody binds to an epitope in a fashion that does not prevent the antifreeze from binding to the ice it will form a complex which can block a greater surface area. This effect would be further enhanced if the lgG binds two antifreeze proteins and Ibereby blocks the ice surface between the two proteins. In addition the antifreeze-antibody complex should project out from the surface of the ice further than the antifreeze alone. Therefore if eventual ice growth in the presence of antifreeze protein should occur as the high radius of curvature growthfronts (Fig. 4b) overgrow the antifreeze and fuse, then the antifreeze-antibody complexes (Fg. 4c) should inhibit such growth and enhance the thermal hysteresis. An extension of this argument suggests that the addition of goat anti-rabbit IgG to the antifreeze-rabbit IgG complexes, thus forming very large antifreeze + anti-antifreeze IgG + anti-IgG complexes (Fig. 4d), would be expected to even further increase activity. As shown in Table I, this was the case. Data on fish glycopeptide antifreezes which demonstrate a direct relationship between the size of the glycopeptide and its activity support this general idea [24]. An alternative to the above explanation for the antibody enhancement effect is that the binding of antibody to the antifreeze protein may change the conformation of the antifreeze protein, or produce some other effect, such that the ice bin~ing abilities of the antifreeze are increased. Such an explanation is unlikely, since this would suggest that the native antifreeze protein is not in its optimal form. Also, the further increase in activity resulting from the addition of the goat anti-rabbit IgG to the antifreeze-antibody complex is not explained by induction of a conformational or other change in the antifreeze protein by addition of the second antibody. However, the experiments with the Fab fragments prepared from the anti-Tenebrio antifreeze tgG wer~ performed to clarify this possibility. The Fab fraga, ents, since they contain the antigen binding site of the lgG, do bind to the antifreeze protein but they failed to increase activity. This result is contrary to the suggestion that the enhancement of antifreeze protein activity occurs because the antibody causes a conformational change in the antifreeze which enhances binding. Because the Fab fragments are ~- 50 kDa, some enhancement of activity might be expected upon their addition to antifreeze protein based on the model proposed in
Fig. 4. Perhaps the spanning of adjacent antifreeze proteins by an intact IgG is required for the effect. As noted in the introduction, purified Dendroides antifreeze protein, even at very high concentrations, does not produce the 3 - 6 ° C of thermal hysteresis commonly present in the hemolymph of overwintering larvae. Recent studies (Wu and Duman, unpublished data) have shown that certain proteins can activate the Dendroides antifreeze proteins. The results presented here suggest that this activation may result from the binding of these activator proteins to the antifreeze proteins. Acknowledgements This study was supported by National Science Foundation grant DCB-87-09872 to J.G.D. and a grant from the Indiana Academy of Sciences to D.W.W. References 1 DeVries,A.C. (1971) in Fish Physiology(Hoar, W.S.and Randall, DJ., eds.), VoL 6, pp. 157-190. Academic Press, New York. 2 DeVries,A.C. (1983) Annu. Rev. Physiol.45, 245-260. 3 Feaney, R.E. and Barcham,T.S. (1986) Annu. Rev. Biophys.15, 59-78. 4 Davies,P.I., Hew,C.L. and Fletcher, G.L. (1988)Can. J. Zool.66, 2611-2617. 5 Davies, P.C. and Hew, C.L. (1990) FASEBJ. 4, 2460-2468. 6 Raymond, J.A. and DeVries, A.L. (1977) Proc. Natl. Acad. Sci. USA 74, 2589-2593. 7 Raymond, J.A., Wilson, P. and DeVties, A.L. (1989) Proc. Natl. Acad. Sci. USA 86, 881-885. 8 Duman, J.G., Xu, L., Neven, L.G., Tursman, D. and Wu, D.W. (1990) in Insectsat Lov, Temperature(Lee, ILE. and Denlinger, D.L., eds.), in press. 9 Duman, J.G. (1979) J. Comp. Physiol. 131, 347-352. 10 Block, W. and Duman,J.O. (1989) J. Exp. Zool. 250, 229-231. ll Patterson, £L. and Duman, J.G. (1979) J. Exp. Zool. 2104, 361367. 12 Patterson,J.L. and Doman,J.G. (1982)J. Exp.Zool.219, 381-384. 13 Patterson, J.C., Kelly, TJ. and Duman, J.G. (1981) J. Comp. Physiol. 142, 539-542. 14 Tomehaney, A.P., Morris, J.P., Kang, S.H. and Dum~.~q.,J.G. (1982) B;.cchewdstry21, 7i6-721. 15 Hew, C.L., Kao, M.H. and So, Y.P. (1983) Can. J. ZooL 61, 2324-2328. 16 Schneppenheim,R. and Thee.de, H. (1980)Comp. Biochem. PhysioL B. 67, 561-568. 17 Duman,J.G. (1979) J. InsectPhysiol.25, 805-810. 18 Duman,J.G. (1980) J. Comp. Physiol. 136, 53-59. 19 Daman,J.G. (1984) J. Exp. Zool. 230, 355-361. 20 Xu, L. and Duman,J.G., J. Exp. Zool., in press. 21 Engvall,E. and Perlman, P. (1972) J. lmmunol. 109, 129-135. 22 Porter, R. (1963) in Psotidesin Biological Fluids(Peeters, H., ed.), pp. 11-15, Elsevier, Amsterdam. 23 DeVries,A.L. (1986) Methods Enzymol.127, 293-303. 24 Schrag, J.D., O'Grady, S.M. and DeVries, A.L. (1982) Biochim. Biophys.Acta 717, 322-326.
Biochimica et Biophysica Acta~ 1076(1991) 416-420 © 1991 Elsevier Science Publishers B.V. 0167..4838/91/$03.50 ADONIS 0167483891001I4A
BBAPRO 33844
Enhancement of insect antifreeze protein activity by antibodies D i n g W e n W u , J o h n G , D u m a n a n d Lei X u Department of Biological Sciences, University of Notre Dame, Notre Dame, IN (U.S.A.) (Received 13 July 1990) (Revised manuscript received 12 October 1990)
Key words: Antifreezeprotein;Coldtolerance;Insectantifreeze;Thermalhysteresisprotein Antifreeze proteins, produced by many cold water marine teicost fish and terrestrial arthropo~ (insects, spiders, etc.), inhibit ice crystal growth by a non-ealligative mechanism, probably by adsorbing onto the surface of potential seed ice crystals and thereby blocking growlh at preferred growth sites, in this study it is demonstrated that the activity of two insect antifreeze proteins is greatly increased by the addition of specMic rabbit polydonal antibodies to the antifreezes. A model is presented which suggests that the enhancement occurs because the antifrceze-antibody complex, being much larger than the antifreeze protein alone (a minimal 7-8-fold increase in size), blocks a larger area of the ice crystal surface and extends fmltber above the. sm'face, thus requiring the temperahn-e to be further lowered before e~stal grog~h proceeds. This idea is further supported by the fmding that addition of goat anti-rabbit IgG to the antifreeze protein + anti-antlfreeze protein antibody complexes further enhanced activity. lntrodnefian Antifreeze proteins have been best studied in cold water marine teleost fishes. (for a review see Refs. i-3). These antifreezes inhibit ice crystal growth by a noncolligative mechanism whereby the freezing point is depressed below the melting point, thus producing a so-called thermal hysteresis. Evidence indicates that the proteins exert their antifreeze effect by an adsorptioninhibition mechanism in which they adsorb onto the surface of potential seed ice crystals, the-eby inhibiting crystal growth [5-7]. Purified antifreeze proteins demonstrate a hyperbofic relationship between protein concentration and thermal hysteresis activity, with the plateau of activity for the more active forms of fish antifreeze proteins at approx. 1.5 o C. Thus higher levels of the antifreeze provide sufficient freezing point depression, in combination with the colligative depression resulting primarily from inorganic ions, to lower the freezing point of the body fluids of the fish below that of seawater. Antifreeze proteins are also known to be produced by terrestrial arthropods, including many insects [8]. spiders [9], a centipede and an Antarctic mite [10] and they have been purified from four species of insects [11-16]. These include the antifreeze proteins used in Correspondence:J. D,,man; Dcpamnentof BiologicalSciences,Universityof Notre Dame.Notre Dame,1N 46556,U.S.A.
this study, from the larvae of the beetles Tenebrio molitor and Dendroides canadensis. The relationship between activity and concentJation for Tenebrio antifreeze proteins is similar to that of the fish [17,18]. Purified Dendroides antifreeze proteins are more active, with
maximal hysteresis activity of purified protein at -~ 2.5°C [8], however mid-winter mean hemolymph hysteresis values of Dendroides populations are typically 3 to 6 ° C with some individuals having 8 - 9 ° C [19]. Recent work OVu and Duman~ unpublished data) has shown that certain proteins can activate the purified Dendroides antifreeze proteins to produce levels of hysteresis comparable to those seen in the winter hemolymph. In the study presented here we show that specific antibodies to insect antifreeze proteins also increase their hysteresis activity. Materials and Methods Preparation of antiserum. Larval Dendroides canadensis were collected from partially decompo,'ed logs in
woodlots near South Bend, IN. Antifreeze proteins were purified OVu, Duman, Cheng and Castellino, unpublished data), and a fraction containing a mixture of two antifreeze proteins (H1 and H2) were used to raise antiserum in male New Zealand white rabbits [20]. (Note that Dendroides hemolymph contains four similar/mmunological!y indis'dnct antifreeze proteins.) Tenebrio molitor, originally purchased from Connecticut Valley Biological Supply, were cultured in the
417 laboratory on a diet of wheat bran. Larger larvae were periodically acclimated at 10*C and a photoperiod of 8 light/16 dark to induce antifreeze protein production. Antifreeze proteins were purified [18] and used to raise antiserum in male New Zealand white rabbits (Xu, Duman and Goodman, unpublished data). ELISAs were used to determine the antibody titres of the two antisera [21]. Peroxidase conjugated goat antirabbit IgG antiserum (Calbiochem) was used as the second antibody for color development. The final detectable concentration of anti-Dendroides antifreeze protein antiserum was a 2TM dilution, and that of the antiTenebrio antifreeze protein antiserum was a 215 dilution. Preparation of IgG and Fab fragments. Total IgG was prepared from the rabbit Tenebrio antifreeze antiserum using an AFFINICA protein A / G column (Schleicher and Schuell) with a binding buffer of 1.5 M glycine, 3 M NaC (pH 8.9) and an elution buffer of 0.2 M glycine-HCl (pH 2.5) at a flow rate of 0.5 ml/min. Fab fragments [22] of the IgG were prepared by digesting the IgG with papain for 10 h (10/tg papain/mg IgG) at 37°C in a buffer of 100 mM sodium acetate, 50 mM cysteine and 1 mM EDTA (pH 5.5). Digestion was terminated by adding 75 mM iodoacetamide. Thc preparation was dialyzed against the binding buffer as used for IgG preparation and the Fab fragments were purified using an AFFINICA protein A / G column as described ::hove. Both the IgG and Fab fragments ran as single bands on SDS-PAGE (non-reducing conditions), indicating their purity. An ELISA (as described above) was used to determine the titre of the purified IgG. Beginning with an IgG concentration of 10 mg/ml, a 2-fold dilution series was run. The end point was 217. Effect of antibodies on antifreeze protein activity. Thermal hysteresis antifreeze activity was determined using the freezing point-melting point difference (thermal hysteresis) technique [23]. To determine the effect of the antibodies on antifreeze protein activity, the thermal hysteresis of the antifreeze proteins was determined at various concentrations, with and without the presence of antisera. Antisera was added to a final concentration of 0.5~ (v/v) in these experiments. Preimmunization rabbit sera was used as a control. To determine the effect of variations in antibody concentration on antifreeze protein activity, Dendroides antifreeze at a final concentration of 2.5 m g / m l was treated with various concentrations of antiserum, ranging in fmal concentration from 0 to 200%. Antiserum concentrations up to 50% were achieved by diluting the antiserum with buffer as appropriate, and then mixing the diluted antiserum 1/1 (v/v) with a 5 m g / m l antifreeze protein solution. For example, to get a final concentration 25% antiserum and 2.5 m g / m l antifreeze, the original antiserum was diluted 1/1 with buffer, then 1 vol. of the diluted antiserum was added to 1 vol. of a
5 m g / m l antifreeze solution. Concentrations of antiserum above 50% were achieved by freeze drying a volume of the antiserum, redissolving the antiserum in a smaller volume of buffer, and then adding this to the antifreeze protein solution as appropriate. For example, to get a 200% final antiserum solution 1 vol. of antiserum was freeze dried, redissolved in 1 / 4 vol. of buffer, and this mixed 1/1 with a 5.0 mg/ml soltion of antifreeze protein. Effects of P A N S O R B I N and goat-anti rabbit IgG on the activity of anti-antifreeze antiserum treated antifreeze protein. PANSORBIN (Staphylococcus aureus cells coated with protein-A, purchased from Calbiochem. Binding capacity: 2.05 rag human l g G / m l of cell suspension.) at a final concentration of 33% was added to an aqueous solution containing Dendroides hemolymph (33%, v/v, final concentration) and anti-Dendroides antifreeze-antiserum (33%, v/v, final concentration). The mixture was incubated at room temperature for 30 rain, centrifuged at 7 0 0 0 × g for 5 rain at 0 ° C and the supernatant removed. Thermal hysteresis activity of the superuatant was determined. Solutions of Dendroides hemolymph diluted to 33% final concentration with Tris-NaCl buffer, and of hemolymph (33%) plus antiserum (33%) were used as controls. The effect of addition of goat-anti rabbit lgG (Calbiochem) on the activity of a mixture of anti-Dendroides antifreeze antiserum plus Dendroides antifreeze protein was determined. Goat-antirabbit antiserum (final concentration of 33%) was added to anti-Dendroides antifreeze antiserum (final concentration of 33%) and Dendroides hemolymph (final concentration of 33%). The mixture was incubated at room temperature for 30 rain and the activity determined. Controls were diluted Dendroides hemolymph (33%) and Dendroides hemolymph (33%) plus antiserum (33%). Effects of anti-Tenebrio antifreeze-lgG and Fab fragments on Tenebrio antifreeze protein activity. Purified Tenebrio antifreeze (final concentration of 10 mg/ml) was treated with either lgG (final concentration: 5 mg/ml) prepared from anti-Tenebrio antifreeze antiserum (as described above) or purified Fab fragments (final concentration: 3 mg/ml) from the lgG and the thermal hysteresis activity was determined. The control was Tenebrio antifreeze protein (10 mg/ml) alone. Statistics. One-way analysis of variance (ANOVA), and in some eases multiple comparisons tests frukey's) were used to analyze the data for statistical significance. R ~
Enhancement of AFP activl.O, by an:ibodies. Contrary to our expectations, when anti-Dendroides antifreeze antiserum was added to the aqueous solution of Dendroides antifreeze protein, the thermal hysteresis activity increased (Fig. 1). Note the approximate doubling of
418 4-
¢J
3.0 •
2.o.
i
-r =
J
2.
J= on
1.0'
J 0.0'
1'0
2'0
0.0
3'0
Fig. l. Enhancing effect of addition of anti-Dendroides antifreeze antiserum (final volume of 0.5~, v/v) on the thermal hysteresis antifreeze activity of Dendroides antifreeze protein. ~ activity at various concentration~ of antifreeze protein in distilled water; ×, activity at various concentrations of antifreeze protein with addition of 0.5~ prcimmouized rabbit serum; and A, activity at various concentrations of antifree=e protein with addition of 0.5~ anti.Dendroides antifreeze protein antiserum. Addition of antiserum caused a statistically significant increase in activity at all concentrations tested (P < 0.05; one-wayANOVA).
activity with addition of antibody at antifreeze concentrations above - 3 m g / m l and the presence of activity at very low antifreeze protein concentrations where without antibody addition there was none. The slight increase in activity resulting from addition of preimmunization rabbit serum is probably due to the addition of salts in the serum to the antifreeze protein in distilled H zO, an effect we have previously recognized. (Note that the preimmunization serum does not have hysteresis activity.) To determine whether the antigen-antibody complex was soluble, Dendroides hemolymph (containing antifreeze protein) was added to an equal voL of anti-Dendroides antifreeze antiserum and the mixture was centrifuged at 6 0 0 0 × 8 ( 0 ° C ) for 15 min. Precipitate was not visible and thermal hysteresis activity was not lost. An even more exaggerated effect was observed with addition of anti-Tenebrio antifreeze antiserum to Tenebrio antifreeze protein (Fig. 2). The hysteresis activity at an antifreeze protein concentration of 2 m g / m l increased nearly 5-fotd. T h e effect on thermal hysteresis of addition of different ratios of antibody to antifreeze protein was investigated by varying the percentage of anti-Dendroides antifreeze antiserum added to a 5 m g / m l concentration of Dendroides antifreeze proteht (Fig. 3). An antiserum concentration of 0.02~% had no effect on activity, howe:'er, 0.05 and 0.50% antiserum caused a statistically significant increase (Tukey's). Interestingly, higher antiserum levels caused a significant decrease in activity as compared to that at 0.50%, and did not result in a significant, increase in activity above that without antiserum. Consequently, only lower ratios of a n t i b o d y / antifreeze produced the enhancement. Although the ex-
210
1.0
AFP (mg/ml)
AFP (mg/ml)
Fig. 2. Enhancing effect of addition of anti-Tenebrio antifreeze antiserum (final volume of 0.5~. v/v) on the thermal hysteresisantifreeze activity of Tenebrio antifreeze proteins. [3, activity at various concentrations of Tenebrio antifreeze protein either in distilled water or with addition of 0.5~ (v/v of final volume) prcimmanized rabbit serum; and a, activity at various concentrations of antifree-..eprotein with addition of 0.5~ (v/v of final volume) anti-Tenebrio antifreeze protein antiserum. Addition of antiserum caused a statistically significant increase in activity at all concentrationstested (P < 0.05; one-way ANOVA).
planation for the lack of enhancement at higher antibody/antifreeze ratios is unknown to us, one possibility is that at higher ratios more than one antibody molecule may bind each antifreeze molecule and thus produce a steric hindrance which precludes adsorption of the antifreeze protein to ice. Effects of PANSORBIN, anti-lgG and Fab fragments on activity. As shown in Table I, addition of PANSORBIN to a solution containing Dendroides antifreeze protein (hemolymph) and antifreeze antiserum eliminated the hysteresis activity. The protein-A coating the bacterial cells should, and apparently did, bind the antifreeze protein antibody complexes. The cells were then centrifuged out, titus removing the activity. (Notc, activity disappeared upon addition of PANSORBIN, even prior to centrifugation.)
°i 3: t-
O 0.0
0.25
0.5
50
Concentration
100
150
200
of A S (%)
Fig. 3. Effect of addition of various concentratioqs (~, v/v, of final volume) of anfi-Dendroides antifreeze protein antiserum on the thermal hysteresisantifreeze activity of Dendroidesantifreeze protein (2.5 mg/ml, final concentration). Concentrations of antiserum below 0.55 were most effective.
419 TABLE I Effects of PANSORBIN, anti-lg6 and Fab fragments on activity
(A) Effect of addition of PANSORBIN or goat anti-rabbit lgG antibodies on the thermal hysteresis antifreeze activity of diluted (33%) Dendroides hemolymphtreated with anti-Dendroides antifreeze antiserum. (B) Effects of addition of IgG from anti-Tenebrio antifreeze antiserum, or Fah fragments from this IgG, to Tenebrio antifreeze protein. Values reported are means:i:S.D. Values were compared using one-wayANOVA.WheneverANOVA proved significant, further analysis was conducted using Tukey's multiple comparisons test. Samplenmans having the same Tukey's rank are not significantly different from one another, but are significantlydifferent from sample means with different Tukey's ranks. Sample (A) 33% Dendroides hemolymph 33% hemolymph+33% antiserum 33% hemolymph+ 33% antiserum + PANSORBIN 33% hemolymph+ 33% antiserum + anti-lgG antiserum (B) Tenebrio antifreeze protein (10 mg/ral) Tenebrio antifreeze protein + lgG (5 mg/rul) Tenebrio antifreeze protein + Fab fragments(3 mg/ml)
growth front proceeds as a circular plate as shown in Fig. 4a, Antifreeze proteins inhibit this preferred growth by adsorbing onto the ice surface, thereby increasing the radius of curvature of the growth fronts (Fig. 4b), raising the interracial free energy and requiring a greater amount of undercoofing to achieve significant growth. Based on this adsorption-inhibition m e c h a n i s m of antifreeze protein activity [6,7], it is likely that the increased activity provided by the addition of antibody to produce soluble antifreeze-antibody complexes is due to
TH ( o C)
Tukey's rank 1.20:i:0.09 C 1.72+0.08 B
0.00+0.00
D
2.86+0.10
A
1.705:0.20
b
2.725:0.51
a
1.71 + 0.70
b
Addition of goat anti-rabbit lgG antiserum to the solution of antifreeze protein and anti-antifreeze antiserum (containing the antifreeze protein + antiantifreeze protein I g G complex) further increased activity (Table I), presumably due to formation of the antifreeze protein + anti-antifreeze protein lgG + anti-lgG complex. As expected, addition of total IgG from anti-Tenebrio antifreeze antiserum to Tenebrio antifreeze protein increased activity (Table I). However, addition of purified Fab fragments from this same lgG did not affect the activity. (The Fab fragments of the IgG contain the antigen binding sites.)
Discussion The addition of specific polyclonal antibodies enhanced the thermal hysteresis antifreeze activity of the Dendroides and Tenebrio antifreeze proteins at all antifreeze concentrations tested. As expected the addition of PANSORBIN to the antifreeze-antibody solutions removed all activity, demonstrating that the antifreezeantibody complexes were present. In contrast, addition of goat anti-rabbit I g G antiserum to solutions containing antifreeze-antibody complexes resulted in a further increase in activity. A model which attempts to explain the results is shown in Fig. 4. Normally ice crystals preferentially grow perpendicular to their C axis by addition of water molecules, probably to step sites. The
surface o f an i c e c r y s t a l
m
growth of the ice crystal antifreezeprotein
d~N
anti-AFP IgG
~II~,
anti-lgG antibody
Fig. 4. Diagrammatic representation of the adsorption - inhibition model of antifreeze protein activity with an elaboration thereon m explain the antibody enhancement effect. (a) Normal growth of ice (antifreeze protein not present) at a step site with the new growth front forming as a circular plate at a temperature just below the melting point. (b) Inhibition of growth by adsorbed antifreeze proteins. This increases the radius of curvature of the growth fronts and requires a decrease in temperature for significant growth to occur. Antifreeze proteins may eventually be overgrown,perhaps leading to rapid growth unless inhibited by further adsorption of antifreeze proteins. (c) Additional inhibition of crystal growth is provided by adsorption of antifreeze protein to which anti-antifrecee protein 18G is attached. At the low ratios of antibody/antifrceze protein used in these studies most antibody should hind two antifreeze proteins, as shown here (d) Even greater inhibition of growth results from adsorption of antifreeze protein plus anti-antifreezeprotein |gG plus anti-lgG antibody complexes.
420 enhanced blocking of potential growth sites by these complexes (Fig. 4c). The antibody, mainly IgG, is much larger (--- 150 kDa) than the antifreeze proteins (15-22 kDa), and one IgG can bind a maximum of two antifreeze proteins. (At the low antibody/antifreeze ratios generally used in this study most of the antibody molecules are expected to bind two antifreeze protein molecules.) Thus, if the antibody binds to an epitope in a fashion that does not prevent the antifreeze from binding to the ice it will form a complex which can block a greater surface area. This effect would be further enhanced if the lgG binds two antifreeze proteins and Ibereby blocks the ice surface between the two proteins. In addition the antifreeze-antibody complex should project out from the surface of the ice further than the antifreeze alone. Therefore if eventual ice growth in the presence of antifreeze protein should occur as the high radius of curvature growthfronts (Fig. 4b) overgrow the antifreeze and fuse, then the antifreeze-antibody complexes (Fg. 4c) should inhibit such growth and enhance the thermal hysteresis. An extension of this argument suggests that the addition of goat anti-rabbit IgG to the antifreeze-rabbit IgG complexes, thus forming very large antifreeze + anti-antifreeze IgG + anti-IgG complexes (Fig. 4d), would be expected to even further increase activity. As shown in Table I, this was the case. Data on fish glycopeptide antifreezes which demonstrate a direct relationship between the size of the glycopeptide and its activity support this general idea [24]. An alternative to the above explanation for the antibody enhancement effect is that the binding of antibody to the antifreeze protein may change the conformation of the antifreeze protein, or produce some other effect, such that the ice bin~ing abilities of the antifreeze are increased. Such an explanation is unlikely, since this would suggest that the native antifreeze protein is not in its optimal form. Also, the further increase in activity resulting from the addition of the goat anti-rabbit IgG to the antifreeze-antibody complex is not explained by induction of a conformational or other change in the antifreeze protein by addition of the second antibody. However, the experiments with the Fab fragments prepared from the anti-Tenebrio antifreeze tgG wer~ performed to clarify this possibility. The Fab fraga, ents, since they contain the antigen binding site of the lgG, do bind to the antifreeze protein but they failed to increase activity. This result is contrary to the suggestion that the enhancement of antifreeze protein activity occurs because the antibody causes a conformational change in the antifreeze which enhances binding. Because the Fab fragments are ~- 50 kDa, some enhancement of activity might be expected upon their addition to antifreeze protein based on the model proposed in
Fig. 4. Perhaps the spanning of adjacent antifreeze proteins by an intact IgG is required for the effect. As noted in the introduction, purified Dendroides antifreeze protein, even at very high concentrations, does not produce the 3 - 6 ° C of thermal hysteresis commonly present in the hemolymph of overwintering larvae. Recent studies (Wu and Duman, unpublished data) have shown that certain proteins can activate the Dendroides antifreeze proteins. The results presented here suggest that this activation may result from the binding of these activator proteins to the antifreeze proteins. Acknowledgements This study was supported by National Science Foundation grant DCB-87-09872 to J.G.D. and a grant from the Indiana Academy of Sciences to D.W.W. References 1 DeVries,A.C. (1971) in Fish Physiology(Hoar, W.S.and Randall, DJ., eds.), VoL 6, pp. 157-190. Academic Press, New York. 2 DeVries,A.C. (1983) Annu. Rev. Physiol.45, 245-260. 3 Feaney, R.E. and Barcham,T.S. (1986) Annu. Rev. Biophys.15, 59-78. 4 Davies,P.I., Hew,C.L. and Fletcher, G.L. (1988)Can. J. Zool.66, 2611-2617. 5 Davies, P.C. and Hew, C.L. (1990) FASEBJ. 4, 2460-2468. 6 Raymond, J.A. and DeVries, A.L. (1977) Proc. Natl. Acad. Sci. USA 74, 2589-2593. 7 Raymond, J.A., Wilson, P. and DeVties, A.L. (1989) Proc. Natl. Acad. Sci. USA 86, 881-885. 8 Duman, J.G., Xu, L., Neven, L.G., Tursman, D. and Wu, D.W. (1990) in Insectsat Lov, Temperature(Lee, ILE. and Denlinger, D.L., eds.), in press. 9 Duman, J.G. (1979) J. Comp. Physiol. 131, 347-352. 10 Block, W. and Duman,J.O. (1989) J. Exp. Zool. 250, 229-231. ll Patterson, £L. and Duman, J.G. (1979) J. Exp. Zool. 2104, 361367. 12 Patterson,J.L. and Doman,J.G. (1982)J. Exp.Zool.219, 381-384. 13 Patterson, J.C., Kelly, TJ. and Duman, J.G. (1981) J. Comp. Physiol. 142, 539-542. 14 Tomehaney, A.P., Morris, J.P., Kang, S.H. and Dum~.~q.,J.G. (1982) B;.cchewdstry21, 7i6-721. 15 Hew, C.L., Kao, M.H. and So, Y.P. (1983) Can. J. ZooL 61, 2324-2328. 16 Schneppenheim,R. and Thee.de, H. (1980)Comp. Biochem. PhysioL B. 67, 561-568. 17 Duman,J.G. (1979) J. InsectPhysiol.25, 805-810. 18 Duman,J.G. (1980) J. Comp. Physiol. 136, 53-59. 19 Daman,J.G. (1984) J. Exp. Zool. 230, 355-361. 20 Xu, L. and Duman,J.G., J. Exp. Zool., in press. 21 Engvall,E. and Perlman, P. (1972) J. lmmunol. 109, 129-135. 22 Porter, R. (1963) in Psotidesin Biological Fluids(Peeters, H., ed.), pp. 11-15, Elsevier, Amsterdam. 23 DeVries,A.L. (1986) Methods Enzymol.127, 293-303. 24 Schrag, J.D., O'Grady, S.M. and DeVries, A.L. (1982) Biochim. Biophys.Acta 717, 322-326.
