Relationship of amino acid composition and molecular weight of antifreeze glycopeptides to non-colligative freezing point depression

Biochimica et Biophysica Acta, 717 (1982) 322-326

322

Elsevier Biomedical Press BBA21195

RELATIONSHIP OF AMINO ACID COMPOSITION AND MOLECULAR WEIGHT OF ANTIFREEZE GLYCOPEPTIDES TO NON-COLLIGATIVE FREEZING POINT DEPRESSION JOSEPH D. SCHRAG, SCOTT M. O'GRADY and ARTHUR L. DEVRIES

Department of Physiology and Biophysics, 524 Burrill Hall, University of Illinois, Urbana, IL 61801 (U.S.A.) (Received February 2nd, 1982)

Key words: Glycopeptide; Antifreeze glycopeptide; Amino acid composition; Molecular weight; Freezing point depression

Many polar fishes synthesize a group of eight glycopoptides that exhibit a non-coHigative lowering of the freezing point of water. These glycopeptides range in molecular weight between 2600 and 33 700. The largest glycopeptides [1-5] lower the freezing point more than the small ones on a weight basis and contain only two amino acids, alanine and threonine, with the disaccharide galactuse-N-acetyl-galactosamine attached to threonine. The smaller glycopeptides, 6, 7, and 8, also lower the freezing point and contain proline, which periodically substitutes for alanine. Glycopeptides with similar antifreeze properties isolated from the saffron cod and the Atlantic tomcod contain an additional amino acid, arginine, which substitutes for threonine in glycopeptlde 6. In this study we address the question of whether differences in amino acid composition or molecular weight between large and small glycopeptides are responsible for the reduced freezing point depressing capability of the low molecular weight glycopeptides. The results indicate that the degree of amino acid substitutions that occur in glycopeptid~ 6 - 8 de not have a significant effect on the unusual freezing point lowering and that the observed decrease in freezing point depression with smaller glycopeptides can be accounted for on the basis of molecular weight.

Introduction Many polar fishes which inhabit ice-laden sea water synthesize macromolecular 'antifreezes' which lower the freezing point of their body fluids below the freezing point of sea water (-1.9°C) [1-3]. The freezing point is lowered in a non-colligative manner. The freezing point depression is believed to result from the adsorption of antifreeze molecules to the surface of ice and this depression is not dependent upon the number of particles in solution [4,5]. The freezing point of serum is 1.51.7°C lower than the melting point. The melting point of ice in antifreeze solutions is what would be predicted on the basis of colligative relationships. Most Antarctic and several Arctic fishes pro-

0304-4165/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

duce glycopeptide antifreezes [1,6-8]. Eight glycopeptides ranging in size from 2600 to 33700 daltons have been isolated [9,10]. They are composed of the repeating glycotripeptide: Ala-Ala-Thr ~ o ~ disaccharide where the disaccharide is fl-D-galactopyranosyl-(1 3)-2-acetamido-2-deoxy-a-D-galactopyranose [1 l-14]. The small glycopeptides occasionally have proline substituted for alanine at position one of the glycotripeptide [12,15]. The saffron cod (Eleginus gracilis) and Atlantic tomcod (Microgadus tomcod) produce a glycopeptide in which arginine is also occassionally substituted for threonine [6,16].

323 The larger glycopeptides (numbered 1 to 5 according to electrophoretic mobilities) exhibit more antifreeze activity than the low molecular weight glycopeptides (numbered 6-8). Antifreeze activity appears to result from the adsorption of the glycopeptide to the surface of an ice crystal [5]. Adsorption of the antifreeze increases the curvature of the growth step and inhibits its growth. The greater antifreeze activity exhibited by the larger glycopeptides appears to result from more efficient adsorption to ice [5]. The efficiency of adsorption of the various sizes of antifreeze may be related to two factors. First, the adsorption may be a function of the molecular weight of the glycopeptide. Secondly, adsorption may be dependent upon a match of repeat spacings in the glycopeptides with the spacing of atoms in the ice lattice [3]. The substitution of proline in the small glycopeptides may alter the secondary structure of the antifreeze in a manner which disrupts the repeat spacing and, therefore, interferes with adsorption. It is also possible that a combination of the two factors is involved in the reduced efficiency of the small glycopeptides. In this study, we attempt to determine the effects of molecular size and antino acid compositions on the non-colligative freezing point lowering of the aqueous solutions of the glycopeptide antifreezes. Materials and Methods

Glycopeptide antifreezes were purified from the serum of the Antarctic cod, Dissostichus mawsoni, and the saffron cod by ion exchange chromatography as previously described [9,16]. Separation of glycopeptide 5 from the larger glycopeptides was achieved by chromatographing a mixture of glycopeptides 1-5 from D. mawsoni on a 1.3 × 172 cm Sephadex G- 100 column. Glycopeptides 1-4 were separated by polyacrylamide gel electrophoresis. Fluorescaminelabelled glycopeptides (1-5) were used as markers. Lyophilized glycopeptides (1-5) from D. mawsoni were resuspended in borate buffer (0.9 M H3BO4, 0.18 M NaOH, pH 8.6) at a concentration of 30 mg/ml. The glycopeptide samples (10 /~1 each) were loaded onto a 10% polyacrylamide gel and run at 10 V / c m for approx. 2.5 h. In those sampies used to mark the location of each band, 5/tl

of fluorescamine (Fluram, Roche Diagnostics) at 4 mg/ml in acetone was added to 20/~1 samples. The gel was sliced to separate the bands as indicated by the marker glycopeptides located at each end of the gel. Each slice was placed in Spectrapore 3 dialysis tubing and the glycopeptides were electrophoresed out of the gel into borate buffer. Each sample was dialyzed and lyophilized. To determine the degree of separation and purity of each glycopeptide band, the lyophilized samples (1 mg each) of the individual glycopeptides were resuspended in borate buffer at 30 mg/ml, reacted with fluorescamine as described above and run on a 10% polyacrylamide gel at 10 V / c m for 2.5 h. Glycopeptide 5 was modified by repetitive Edman degradation [17]. The molecular weight was reduced from 10500 to approximately 7900 by performing 14 Edman turns. This procedure does not affect the remaining disaccharide moieties [18]. Samples of lyophilized glycopeptides were weighed and dissolved in distilled water for determination of freezing points. Molar concentrations of solutions of glycopeptides 2, 5 and 6 were calculated using molecular weights of 28 800, 10500 and 7900, respectively [10]. Glycopeptide 5 after Edman degradation had a molecular weight of 7900. The method described by DeVries [4] was used for freezing point determinations. 2-5 /tl were placed in a 10/~1 capillary tube. The tubes were sealed with mineral oil. A small seed crystal was introduced by spraying the air-liquid interface with a refrigerant spray and the sample was placed in a refrigerated bath where temperature was controlled to within +-0.01°C. The capillaries were viewed with the aid of a microscope equipped with cross polarizer lens. The bath temperature was lowered at the rate of 0.01°C per min until the seed crystal began to grow. The temperature at which the seed crystal began to increase in size was taken as the freezing point. The melting point was taken as the temperature at which the seed crystal melted when the bath temperature was raised slowly. Using this technique, large differences between the freezing and melting points were observed for all solutions of the glycopeptides. This non-colligative lowering of the freezing point has been referred to as 'antifreeze activity' [4].

324

Results and Discussion

1

2

3

4

5

7

6

8

9

i0 W

Table I shows a comparison of amino acid compositions of various antifreeze fractions. In order to assess the effects of amino acid composition on antifreeze activity, glycopeptide 5 was reduced in length by Edman degradation to a size equal to that of glycopeptide 6 isolated from D. mawsoni and E. gracilis. Reduction in size of glycopeptide 5 allowed comparison of the freezing behavior of a series of glycopeptides having approximately the same molecular weight but different amino acid compositions. The shortened glycopeptide 5 is composed only of the basic repeating glycotripeptide. Glycopeptide 6 from D. mawsoni has the same repeating unit and also has proline substituted for some of the alanines. Glycopeptide 6 from the saffron cod has arginine substituted for some threonine in addition to the proline substitutions. The polyacrylamide gel shown in Fig. 1 demonstrates that the molecular size of the three fractions are approximately equal. The borate ions in the running buffer of this gel system complex to the hydroxyl groups of the disaccharide moiety of glycopeptides. This confers a negative charge on the glycopeptide which is dependent on the number of disaccharides present on the molecule and, thus, the migration of the glycopeptide is related to its molecular size. The borate in this system is analogous to SDS in SDS-gel electrophoresis. The negatively charged borate ions also interact with the positively charged arginine residues in the saffron cod glycopeptides. The substitution of arginine for a glycosidically linked threonine, thus,

U

w

SOLVENT FRONT

Fig. 1. Polyacrylamide gel electrophoresis pattern obtained from various glycopeptide antifreeze preparations. (1) Standard (D. mawsoni glycopeptide I-5), (2) D. mawsoni glycopeptide 6, (3) D. mawsoni glycopeptide 7 and 8, (4) E. gracilis GP 6, (5) D. mawsoni GP 2, (6) D. mawsoni glycopeptide 3, (7) 1). mawsoni glycopeptide 4, (8) D. mawsoni glycopeptide 5, (9) D. mawsoni modified glycopeptide 5, (10) standard 1). mawsoni (1-5).

does not impair the separation according to molecular size in these glycopeptides. Fig. 2 is a comparison of the freezing points observed for aqueous solutions of these three glycopeptides. The similarities in the values obtained for the three glycopeptides at each concentration indicates that the amino acid substitutions do not alter the non-colligative freezing point depression. The above data suggest that the observed dif-

1,6

L'

-1.4

-12 1,0

TABLE 1

0.8

AMINO ACID COMPOSITIONS OF ANTIFREEZE GLYCOPEPTIDES

0.6 -0.4

Ala Thr Pro Arg a

D. mawsoni

D. mawsoni

Eleginus gracilis

Glycopeptide 1-5 a

Glycopeptid¢-6

Glycopeptide-6 a

1.00 0.50 -

1.00 0.46 0.14 -

1.00 0.47 0.23 0.10

Values are molar ratios with alanine taken O'Grady et al. [16].

-0.2 0 I

I

I

]

2

3

CONCENTRATION(MM)

as

1.0.

From

Fig. 2. Comparison of the freezing point depressions obtained with aqueous solutions of glycopeptides of equal size but different amino acid composition. A, glycopeptide 6 from D. mawsoni; [2, glycopeptide 6 from E. gracilis; O, modified glycopeptide 5 from D. mawsoni.

325 -i,6 -i,4

-1,2 -1.0

0,8

-0,6 -o,q

-0.2

t

I

I

1

2

3

CONCENTRATION(MM)

Fig. 3. Comparisonof the freezingpoint depressions obtained from aqueous solutions of glycopeptides differing only in molecularsize. A, glycopeptide2 (Mr 28800); O, glycopeptide 5 (Mr 10500); l-q,modifiedglyeopeptide5 (Mr 7900). ferences in antifreeze activity of different size glycopeptides may be entirely due to differences in molecular weight. To assess the effect of molecular weight on antifreeze activity, the large molecular weight glycopeptides were separated by electrophoresis. Fig. 1 demonstrates that the glycopeptides were separated into distinct size classes. Comparison of freezing points as a function of molar concentration in Fig. 3 clearly demonstrates I

[

I

I

I

]

I 16

I 18

I

8

I 22

I

4

20

22

2q

depression

and

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-!,2

-1,0

-0.8

•:

-0.6

-o.q

-0.2

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MOLECULAR WEIGHT x 10 -3

F i g . 4.

Relationship

between

freezing

point

molecular weight. All concentrationsare 1 mM.

that the effectiveness of glycopeptides in depressing the freezing point increases with the size of the molecule. Fig.4 demonstrates the differences in freezing point observed with equimolar concentrations of glycopeptides of the various size classes. The data point at 2000 daltons was adapted from the data of Geoghegan et al. [18] after glycopeptide 8 had been shortened by one glycotripeptide by Edman reaction. This data suggests that glycopeptides smaller than about 2000 daltons exhibit only the freezing point depression expected on the basis of colligative relationships and little or none of the non-coUigative freezing point depression associated with the antifreeze glycopeptides. The observation that the substitution of proline into the peptide backbone does not significantly affect antifreeze activity suggests that either the secondary structure of the glycopeptides involves a conformation which is not disrupted by proline or that antifreeze activity is dependent only on the number of carbohydrate side chains present and not their relative orientation. The secondary structure of the antifreeze glycopeptides is uncertain. Raymond et al. [19] suggest from early CD data that the glycopeptides are random coils. Bush et al. [20], however, suggest that the glycopeptides form left-handed helices. Random coils and lefthanded helices are two conformations which proline residues do not disrupt due to steric considerations [21]. If the secondary structure of the glycopeptides involves either of these conformations, one would not expect the proline substitution to be disruptive and antifreeze activity would not be significantly affected. The data of this study are consistent with either of these conformations. A n o t h e r interesting point is that the substitution of arginine for threonine does not affect antifreeze activity. There is a considerable body of evidence which indicates that the carbohydrate is essential for antifreeze activity of glycopeptide antifreezes [13,22]. Substitution of arginine for threonine reduces the number of disaccharides in the molecule which would lead one to expect a reduced antifreeze activity. Adsorption of antifreeze to ice is thought to occur by hydrogen bonding. Perhaps the arginine residue can replace the disaccharide and participate in hydrogen bond formation with the oxygen in the ice lattice.

326

Another possibility is that the number of glycosidically-linked threonine residues replaced by arginine is small and this may have little effect on the total energy of binding and, hence, show no effect on antifreeze activity. Several conclusions can be drawn from the present study. First, the substitution of proline for alanine in the glycotripeptide has no detectable effect on antifreeze activity. This implies that the secondary structure of the antifreeze glycopeptides involves a conformation which is not disrupted by proline. The substitution of arginine for the glycosidically-linked threonine also has no effect on antifreeze activity. A second important conclusion is that antifreeze activity is a function of the molecular weight of the glycopeptide. The antifreeze activity of glycopeptides 1-5 had previously been considered to be independent of molecular weight [23]. The present study shows, however, that when examined on the basis of molar concentration, the antifreeze activity of all of the size fractions is related to molecular weight.

Acknowledgments This work was supported by the National Science Foundation grants NSF-PCM-77-25166 and NSF-DPP-78-23462 to A.L.D.

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4 DeVries, A.L. (1971) Science 172, 1152-1155 5 Raymond, J.A. and DeVries, A.L. (1977) Proc. NatL. Acad. Sci. U.S.A. 74, 2589-2593 6 Raymond, J.A., Lin, Y. and DeVries, A.L. (1975) J. Exptl. Zool. 193, 125-130 7 Osuga, D.T. and Feeney, R.E. (1978) J. Biol. Chem. 253, 5338-5343 8 VanVoorhies, W.V., Raymond, J.A. and DeVries, A.L. (1978) Physiol. Zool. 51,347-353 9 DeVries, A.L., Komatsu, S.K. and Feeney, R.F. (1970) J. Biol. Chem. 245, 2901-2908 10 DeVries, A.L. and Lin, Y. (1977) in Adaptations Within Antarctic Ecosystems (Llano, G.A., ed.), pp. 439-458 Gulf Publishing Co., Houston 11 DeVries, A.L., Vandanheede, J. and Feeney, R.E. (1971) J. Biol. Chem. 246, 305-309. 12 Lin, Y., Duman, J.G. and DeVries, A.L. (1972) Biochem. Biophys. Res. Commun. 45, 87-92 13 Shier, W.T., Lin, Y. and DeVries, A.L. (1972) Biochim, Biophys. Acta 263, 406-413 14 Shier, W.T., Lin, Y. and Devries, A.L. (1975) FEBS Lett. 54, 135-138 15 Morris, H.R., Thompson, M.R., Osuga, D,T., Ahmed, A.I., Chan, S.M., Vandenheede, J.R. and Feeney, R.E. (1978) J. Biol. Chem. 253, 5155-5162 16 Reference deleted 17 Edman, P. (1970) in Protein Sequence Determination (S. Needleman, ed.), pp. 211-255, Springer-Vedag, Berlin 18 Geoghegan, K.F., Osuga, D.T., Ahmed, A.I., Yeh, Y. and Feeney, R.E. (1980) J. Biol. Chem. 255, 663-666 19 Raymond, J.A., Radding, W. and DeVries, A.L. (1977) Biopolymers 16, 2575-2578. 20 Bush, C.A., Feeney, R.E., Osuga, D.T., Ralapati, S. and Yeh, Y. (1981) Int. J. Peptide Prot. Res. 17, 125-129 21 Cantor, C.R. and Schimmel, P.R. (1980) Biophysical Chemistry, Vol. 1. pp. 95-100, W.H. Freeman and Co., San Francisco 22 DeVries, A.L. (1974) in Biochemical and Biophysical Perspectives in Marine Biology (Malins, D.C. and Sargent, J.R., eds.), Vol. l, pp. 289-330 Academic Press, London 23 Raymond, J.A. and DeVries, A.L. (1972) Cryobiol. 9, 541547