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Structure and mode of action of glycoproteins from an antarctic fish
406
BIOCHIMICAET BIOPHYSICAACTA
BBA 36O81 STRUCTURE AND MODE OF ACTION OF GLYCOPROTEINS FROM AN ANTARCTIC F I S H
W. THOMAS SHIER a, YUAN LIN b AND ARTHUR L. DE VRIESb aThe Armand Hammer Center for Cancer Biology, The Salk Institute for Biological Studies, Post Office Box ~8o9, San Diego, Calif. 92rz2 and bphysiological Research Laboratory, Scripps Institution of Oceanography, University of California, San Diego, LaJolla, Calif. 92037 (U.S.A .)
(Received October 7th, 1971)
SUMMARY The determination of the structures of a series of serum glycoproteins from the Antarctic fish T r e m a t o m u s borchgrevinki has been completed. These glycoproteins are present in a range of molecular weights and they consist entirely of repeating units o f - A l a - T h r - A l a - in which threonine bears a disaccharide moiety that we now show to be a fl-D-galactopyranosyl-(I-+4)-2-acetamido-2-deoxy-a-D-galactopyranosyl moiety. To our knowledge this represents the first determination of the complete structure of a glycoprotein. These glycoproteins have been shown to be partly responsible for the unusually low temperatures at which the blood serums of Antarctic fishes freeze. We have prepared a series of derivatives of the glycoproteins in which hydroxyl groups of the carbohydrate moiety have been chemically modified by acetonation, acetylation or oxidation. The freezing point depressing activity of the derivatives has been evaluated and the results suggest that the activity results from an interaction with water or ice that involves many structural features of the glycoprotein molecules.
INTRODUCTION
An increasing number of physiological functions are being attributed to glycoproteins. Most of these glycoproteins have complicated structures, and to our knowledge, in no previous case has the complete structure of a glycoprotein been determined. Relatively little information is available concerning the role played by the carbohydrate side chains in the mode of action of glycoproteins1. A group of glycoproteins has been isolated from the serum of the fish Trematomus borchgrevinki2, ~, which inhabits McMurdo Sound, Antarctica 4. The freezing point of the serum of the fish is --2.o°C, in contrast to the value of --0.5 to --0.8°C in most of the marine fishes 5, and 30% of this freezing point depression is a result of the presence of these glycoproteins*. On a molal basis they are about 200-500 times Biochim. Biophys. Acta, 263 (1972) 4o6-413
GLYCOPROTEINSFROM AN ANTARCTIC FISH
407
more effective than NaC1 in lowering the freezing point of water 3,e. Therefore, we can conclude that their interaction with water involves something other than the normal colligative properties shown by ideal solutes. These glycoproteins in contrast to most glycoproteins, have a very simple composition and a readily detectable activity. They are present in a range of molecular weights, but each is composed of the same repeating unit, -Ala-Thr-Ala -7, in which the threonine residue bears a disaccharide that was shown by Smith degradation s to be D-galactopyranosyl-2-acetamido-2-deoxy-D-galactopyranose. Two uncertainties about the structure of the carbohydrate moieties remained: (I) the nature of the configuration at each of the anomeric carbons, and (2) whether the galactose to N-acetylgalactosamine linkage is 1-->3 or 1-+ 4. Previous studiesS, 9, have shown that the carbohydrate moieties of these glycoproteins are involved in tile freezing point depressing activity. Borate ions, which are known to complex readily with cis-hydroxyl groups on adjacent carbon atoms 1°, destroy the freezing point depressing activity of these glycoproteins9. Acetylation of only 30% of the hydroxyl groups has also been shown to destroy their activity s, as does treatment with sodium periodate s, or removal of the terminal sugar residue of the oligosaccharide side chains by Smith degradation s. The structure of the carbohydrate moieties has been determined in detail, thereby completing the structure determination of this group of glycoproteins. Knowledge of the complete structure of the carbohydrate moieties has permitted specific modification of hydroxyl groups of the carbohydrate moieties in order to determine the role they play in imparting the unique degree of freezing point depressing activity to this group of glycoproteins. MATERIALS AND METHODS The freezing point depressing glycoproteins (I) were isolated from the blood serum of T. borchgrevinki according to the method of De Vries 2 and De Vries et al. 3. All studies were carried out on a sample of the glycoproteins containing the natural mixture of three components with molecular weights of io 500, 17 ooo and 21 500. A sample of acetylated glycoproteins for NMR analysis was prepared according to the method of Komatsu et al. s. The glycodipeptide (II) was prepared from subtilisindigested glycoproteins according to the method of De Vries et al. 7. a-D-Galactosidase 11 (from fig latex) and/~-D-galactosidase (from jack bean meal) were supplied by Dr Y. T. Li of Tulane University, Covington, La. Galactose oxidase (Galactostat Kit) was purchased from Worthington Biochemical Co. Methylated bovine serum albumin and lactose were obtained from Sigma, deuterated solvents from Mallinckrodt, and lactnlose from Nutritional Biochemical Co. Lactal and/~-D-galactopyranosyl-(I-+4)a-methyl-D-mannopyranoside were synthesized by the method of Haworth et al. TM. NMR spectra were obtained on either a Varian Associates HR-22o or a J E O L JNM PSIoo instrument in deuterated solvents. Incubation of glycoproteins with a- and'~-galactosidases A sample of glycoproteins (3 rag) was dissolved in 15o ~ul of 0.05 M sodium acetate buffer (pH 4-5), and to this solution 3o/~1 of enzyme (I unit enzyme per IO #1 ; one unit of enzyme is defined as that amount which hydrolyzes I #mole of p-nitroBiochim. Biophys. Acta, 263 (1972) 4o6-413
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W.T. SHIER et al.
phenyl-a-I)-galactoside or p-nitrophenyl-fl-D-galactoside per min at 25°C) were added. The incubation was carried out at 37°C for 72 h (Y. T. Li, personal communication). After incubation, 20 pl of the reaction mixture was used directly for paper chromatography according to the method of Trevelyan et al. TM. Galactose was used as a standard.
I sopropylidene glycoproteins (III) Glycoproteins (5o rag) were dissolved in a mixture of dimethylformamide (2 ml) and 2,2-dimethoxypropane (I ml). A few crystals of p-toluene sulfonic acid were added as a catalyst and the mixture was stirred at room temperature for 24 h (ref. 14). Sufficient K2CO 3 was added to neutralize the p-toluene sulfonic acid, the salt was removed from the solution by dialysis, and the solution was lyophilized. The NMR spectrum of I I I in ~H20 was similar to that of native glycoproteins, but contained additional absorptions at 6 1.32 p p m to 6 1.54 p p m overlapping with the C-CH 3 absorptions due to alanyl and threonyl residues. Integration of the complex absorption indicated that one isopropylidene group was present for each acetamido group (6 2.00 ppm).
Acetylated isopropylidene glycoproteins (IV) Compound I I I was acetylated according to the method of Komatsu et al. s. The NMR spectrum in E~Hlchloroform contained absorptions at ~ 1.44 to 1.54 p p m (15 H, of which 9 H is due to alanyl and threonyl methyl groups) indicating the retention of a single isopropylidene group, and absorptions at ~ 2.04 p p m (3 H, acetamido group) and at 6 2.12 to 2.25 p p m (12 H, four acetoxyl groups).
Selectively acetylated glycoproteins (V) Compound V was prepared from Compound IV by acid hydrolysis according to the method given below. The NMR spectrum in I2H61dimethylsulfoxide contained no isopropylidene absorption, but contained a complex peak centering at 6 2.Ol p p m (12 H, acetoxyl) and at 6 1.88 ppm (3 H, acetamido).
Aldehydoglycoproteins (VI) Native glycoproteins were oxidized with galactose oxidase according to the method of De Vries 6. Monitoring the reaction indicated oxidation of 1.6 hydroxyl groups per disaccharide residue. The enzymes were separated from the reaction mixture using a DEAE-cellulose column in 0.0025 M Tris buffer (pH 9.6). The aldehydroglycoproteins fraction was collected, dialyzed and lyophilized. Although the NMR spectrum did not show any aldehyde absorptions in the expected region, the product of the reaction reacted like an aldehyde when oxidized with bromine water, and it gave acetylated derivatives (vide infra) that corresponded to oxidation of both sugar residues of the disaccharide moiety. In addition, it migrated at the same rate as native glycoproteins on acrylamide-gel electrophoresis carried out according to the method of De Vries et al. ~, and it did not form an insoluble complex with methylated bovine serum albumin, indicating that no over oxidation to carboxyl groups had occurred. Biochim. Biophys. Acta, 263 (1972) 4o6-413
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Carboxyglycoproteins (VII) Compound VI (4 ° mg) was dissolved in water (2 ml), and CaCO 3 (o.5 g) and bromine (o.o15 ml) was added. The mixture was allowed to stand with occasional shaking for 2 h. The excess Br 2 was destroyed with sodium thiosulfate solution and the mixture was acidified with I.O M HC1 until the excess CaCO 3 dissolved. The mixture was dialyzed and lyophilized to yield 4 ° mg of Compound V I I that forms an insoluble complex with methylated bovine serum albumin and migrates much faster than native glycoproteins on acrylamide-gel electrophoresis.
I sopropylidene aldehydoglycoproteins (VIII) Aldehydoglycoproteins were converted to the isopropylidene derivatives by the method used for the preparation of Compound I I I . The NMR spectrum contained absorptions in the range d 1.32 to 1.54 p p m that integrated for one isopropylidene group per disaccharide residue.
A cetylated isopropylidene aldehydoglycoproteins (IX) Compound V I I I was acetylated according to the method of Komatsu et al.s. The NMR spectrum contained absorptions at ~ 1.22 to 1.53 p p m (complex peak, 15 H, indicating one isopropylidene group per disaccharide residue), ~ 2.00 p p m (3 H, acetamido) and d 2.15 p p m (complex peak, 6 H, acetoxyl).
Selectively acetylated aldehydoglycoproteins ( X ) Compound I X was subjected to mild acid hydrolysis as described below to give Compound X. The NMR spectrum contained no isopropylidene absorptions, but 2.04 p p m (3 H, acetamido), ~ 2.12 p p m (3 H, acetoxyl) and ~ 2.18 p p m (3 H, acetoxyl).
Acid hydrolysis of isopropylidene derivatives Isopropylidene derivatives were mixed with HC1 to give a final concentration of o.oi M, and the mixture was incubated at 95°C for 20 min. The reaction mixture was dialyzed and lyophilized. The NMR spectrum of the products showed no isopropylidene absorptions.
Deacetylation Removal of acetyl groups was carried out in a hydroxylamine solution according to the method of Grossberg and Pressman 15.
Determination of freezing points The freezing point measurements were made with a Fiske Osmometer calibrated with standard NaC1 solutions 3. Since the acetyl and isopropylidene derivatives were not soluble in water, the freezing points of their solutions were determined in i M aqueous dimethylformamide, and compared with the freezing point of solutions of native glycoproteins in the same solvent. RESULTS
Structure of the carbohydrate moiety The NMR spectrum of the native glycoproteins was unsatisfactory for the assignment of the stereochemistry about the two anomeric carbon atoms, since it Biochim. Biophys. Acta, 263 (1972) 4o6-413
41o
w.T. SHIER et al.
H0
~.~ fHzOH HO.v O
/'~ Hb H
CHzOH AcHN~ Ha
O(~HCH, ?H, HtNCHGONHCHCOaH
Fig. I. T h e s t r u c t u r e of t h e glycodipeptide (II) isolated from t h e subtilisin digested glycoproteins. Ha a n d Hb are t h e a n o m e r i c p r o t o n s of t h e G a l N A c a n d Gal residues, respectively. Ac is t h e acetyl group.
contained additional signals in the region of interest. However, the NMR spectrum of the glycodipeptide fragment, II (Fig. I), obtained by subtilisin digestion 7 of the glycoproteins, contained two sharp doublets at $ 4.86 ppm and $ 4.27 ppm, shifted downfield from the other C-H signals and in the region characteristic of anomeric protons 16. The coupling constants of J1,2 - 3 .6 and 7.2 Hz, respectively, suggest one a-linkage and one /~-linkage. Comparison of the chemical shifts of these anomeric protons with those of several reference compounds presented in Table I permits the assignment of a r-configuration to the galactose to N-acetylgalactosamine linkage 17, and hence, an a-configuration to the N-acetylgalactosamine t o threonine linkage. The assignment of a r-linkage to the galactosyl residue was substantiated by the results of the action of a- and ~-galactosidases on the glycoproteins. Only/~-galactosidase was able to release galactose from the glycoproteins. TABLE I NMR
DATA FOR
THE
ANOMERIC
Compound
PROTONS
Anomeric proton of galactosyl residue (ppm)
fl-Gal /%Gal /~-Gal fl-Gal II
(1--+4) (1-+4) (1--+4) (i-~4)
OF GALACTOSE-CONTAINING
Glc Glucal a-Man-OCH 3 Fru
4.23 4.39 4.31 4.43 4.27
DISACCHARIDES
Anomeric proton of other residue
Jz,2 (Hz)
c9 (ppm)
7.2 7 .2 6.8 6.8 7.2
4.99
4.0
-
-
-
4.64 -4.86
.[1,2 (Hz)
-
2. 4 -3.6
An analysis of the NMR spectrum of the acetylated glycoprotein permits the assignment of a I--~4 galactose to N-acetylgalactosamine linkage. In the NMR spectra of acetylated sugars the~absorptions of axial secondary acetoxyl groups can generally be resolved from those of the equatorial secondary acetoxyl groups, and also from acetamido and primary acetoxyl group absorptions in. On acetylation the C-4 hydroxyl~group of the N-acetyl-o-galactosamine residue in the native glycoproteins will give an axial secondary acetoxyl group unless that hydroxyl group is involved in the glycosidic linkage. That is, if the linkage is I---~4, one axial secondary acetoxyl group (on the I)-galactose residue) will be formed, but if the linkage is I --~3, two will be formed. In the NMR spectrum of the acetylated glycoproteins the acetyl Biochim. Biophys. Acta, 263 (1972) 4o6-413
GLYCOPROTEINS FROM AN ANTARCTIC FISH
411
absorptions were partially resolved into the following peaks: 6 1.95 ppm, 3 H, assigned to the acetamido group16; 6 2.03 ppm with a shoulder at ~ 2.08 ppm, 9 H, assigned to the equatorial secondary acetoxyl groups16; a doublet at d 2.15 ppm and 2.17 ppm, 6 H, assigned to the primary acetoxyl groups; and b 2.22 ppm, 3 H, assigned to a secondary axial acetoxyP n. The observed pattern of relative intensities (i.e. 1:3:2 :I) is consistent with a 1-+ 4 linkage, but is inconsistent with a 1-+3 linkage which would require a 1:2:2:2 pattern of relative intensities. Hence, the structure of the disaccharide-threonine moiety is fl-n-galactopyranosyl-(I-+4)-a-2-acetamidodeoxy-n-galactopyranosylthreonine (Fig. I). The freezing point depressing activities of the chemically modified glycoproteins were determined and compared with the activity of the native glycoproteins. The data are summarized in Table II. The formation of isopropylidene derivatives and acetylation did not cause any permanent disruption of the molecule since removal of these groups by mild acid hydrolysis and hydroxylamine, respectively, always regenerated the original activity. TABLE
II
FREEZING
POINT
DEPRESSING
ACTIVITY
OF CHEMICALLY
MODIFIED
GLYCOPROTEINS
R,O
[
R,
o
,,o<.Z-o,/hAo ORs Ac HN
I 0 I -~Ala--Thr--Ala--)- n
Compound
Structure**
I III IV V VI VII VIII IX X
Freezing point depressing activity*
R I = Rs = H, R z = CH,OH (R1) ~ = > C ( C H a ) 2 . R , = C H 2 O H , R a = H (R1) 2 = > C ( C H 3 ) = , R~ = C H 2 O A c , R z = Ac** R x = H , R~ = C H , O A c , R n = A c R 1 = R a = H , R~ = C H O R a = R 3H, R 2 = CO2H (R1) 2 = > C ( C H 3 ) v R~ = C H O , R 3 = H ( R , ) 2 = > C ( C H 3 ) 2, R 2 = C H O , R 3 = A c R~ : H , R~ : C H O , R~ = A c
IOO <5 <5 <5 95 <5 <5 <5 <5
" T h e f r e e z i n g p o i n t d e p r e s s i n g a c t i v i t y o f C o m p o u n d I, n a t i v e g l y c o p r o t e i n s , w a s a s s i g n e d a s i o o . T h e a c t i v i t i e s o f o t h e r c o m p o u n d s w e r e e x p r e s s e d r e l a t i v e t o C o m p o u n d I. "* A c is t h e a c e t v l g r o u p .
DISCUSSION
The structure determined for the disaccharide moieties of the freezing point depressing glycoproteins of T. borchgrevinki shows considerable homology with the oligosaccharide moieties of other glycoproteins. As in all other adequately studied Biochim. Biophys. Ac/a,
263 (1972) 4 o 6 - 4 1 3
412
w.T. SHIER et al.
glycoproteins that contain an N-acetylgalactosamine-threonine linkage, the linkage is of the a-D-anomeric configuration is. The/~-D-galactopyranosyl-(i-+4)-2-acetamido2-deoxygalactopyranose structure that constitutes the disaccharide moieties of freezing point depressing glycoproteins also forms part of a trisaccharide isolated from porcine submaxillary mucin by Katzman and Eylar 19. The disaccharide may also form part of the oligosaccharide of M blood-group substance s°. It has been reported previouslyS,9 that the carbohydrate moieties are essential for the activity of the freezing point depressing glycoproteins. The results presented in this report support and extend this conclusion. The observation that most of the modified glycoproteins are inactive implies that most of the functional groups in the disaccharide moieties are involved in the interaction with water. Several plausible mechanisms by which the glycoproteins may depress the freezing point of aqueous solutions have been put forward 9. The results presented in this report do not permit one to distinguish between models in which depression of the freezing point results from the structuring of water by hydrogen bonding or by inhibition of ice crystal growth through surface adsorption. If a hypothesis featuring water structuring by means of hydrogen bonding is correct, it would appear that the freezing point depressing activity results from a rather specific structuring of water, rather than merely the amount of water ordered, because the carboxyl groups of the inactive carboxyglycoproteins would be expected to order more water in their hydration spheres than aldehyde or hydroxymethyl groups of the active aldehydo- and native glycoproteins, respectively, could order by hydrogen bonding. However, it is difficult to reconcile a water structuring hypothesis with the thermal hysteresis observed in the freezing and melting behavior of solutions of the glycoproteins9. The thermal hysteresis is better explained by the model where the glycoproteins bind to the surface of crystals preventing ice formation and hence lowering of the freezing point. Experiments are in progress to determine what other features of the structure of these glycoproteins are responsible for their freezing point depressing activity. ACKNOWLEDGMENTS
This work was supported in part by National Science Foundation Grant GV27327, Dernham Junior Fellowship J-I58 from the American Cancer Society, California Division (to W.T.S.), and National Institutes of Health Postdoctoral Fellowship I FO2HE5o4o5-oI (to Y.L.). We thank Dr John Wright of the University of California and Mr R. Kaiser of the Salk Institute for obtaining the NMR spectra, and Dr Y. T. Li of Tulane University for kindly supplying a- and fi-galactosidases. REFERENCES
R. G. Spiro, Annu. Rev. Biochem., 39 (197 o) 599. A. L. DeVries, P h . D . Thesis, Stanford University, 1968. A. L. DeVries, S. K. K o l n a t s u and R. E. Feeney, J. Biol. Chem., 245 (197 o) 29Ol. A. L. DeVries and D. E. Wohlschlag, Science, 163 (1969) lO73. V. S. Black, Univ. Toronto Biol. Ser., 59 (1951) 53. A. L. DeVries, in W. H. H o a r and D. J. Randall, Fish Physiol., Vol. 6, Academic Press, New York, I971, p. 157. 7 A. L. DeVries, J. Vanderheede and R. E. Feeney, J. Biol. Chem., 246 (1971) 305 . 8 S. K. K o m a t s u , A. L. DeVries and R. E. Feeney, J. Biol. Chem., 245 (197o) 29o9.
I 2 3 4 5 6
Biochim. Biophys. Aota, 263 (1972) 4o6-413
GLYCOPROTEINS FROM AN ANTARCTIC FISH
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9 A. L. DeVries, Science, 172 (1971) 1151. i o C. A. Zittle, Adv. Enzymol., 12 (1952) 439i i S. I. Hakomori, B. Siddiqui, T.-Y. Li a n d S.-C. Li and C. G. Hellerqvist, J. Biol. Chem., 246 (1971 ) 2271. 12 W. N. H a w o r t h , E. L. Hirst, M. M. T. P l a n t and R. J. W. Reynolds, J. Chem. Soc., (193 o) 2644. 13 W. E. Trevelyan, D. P. P r o c t e r a n d J. S. Harrison, Science, 166 (195 o) 444. 14 M. E. E v a n s , F. W. P a r r i s h and L. Long, Jr., Carbohydr. Res., 3 (1967) 453. 15 A. L. Grossberg and D. P r e s s m a n , Biochemistry, 2 (1963) 90. 16 L. D. Hall, Advan. Carbohydr. Chem., 19 (1964) 51. 17 J. M. VAN DER VEEN, J. Org. Chem., 28 (1963) 364 . 18 R. D. Marshall a n d A. Neuberger, Adv. Carbohydr. Chem. Biochem., 25 (197 o) 4o7 • 19 R. L. K a t z m a n and E. H. Eylar, Biochem. Biophys. Res. Commun., 23 (1966) 769 . 20 D. B. T h o m a s and R. J. Winzler, Biochem. Biophys. Res. Commun., 35 (1969) 811.
Biochim. Biophys. Acta, 263 (1972) 4o6-413
BIOCHIMICAET BIOPHYSICAACTA
BBA 36O81 STRUCTURE AND MODE OF ACTION OF GLYCOPROTEINS FROM AN ANTARCTIC F I S H
W. THOMAS SHIER a, YUAN LIN b AND ARTHUR L. DE VRIESb aThe Armand Hammer Center for Cancer Biology, The Salk Institute for Biological Studies, Post Office Box ~8o9, San Diego, Calif. 92rz2 and bphysiological Research Laboratory, Scripps Institution of Oceanography, University of California, San Diego, LaJolla, Calif. 92037 (U.S.A .)
(Received October 7th, 1971)
SUMMARY The determination of the structures of a series of serum glycoproteins from the Antarctic fish T r e m a t o m u s borchgrevinki has been completed. These glycoproteins are present in a range of molecular weights and they consist entirely of repeating units o f - A l a - T h r - A l a - in which threonine bears a disaccharide moiety that we now show to be a fl-D-galactopyranosyl-(I-+4)-2-acetamido-2-deoxy-a-D-galactopyranosyl moiety. To our knowledge this represents the first determination of the complete structure of a glycoprotein. These glycoproteins have been shown to be partly responsible for the unusually low temperatures at which the blood serums of Antarctic fishes freeze. We have prepared a series of derivatives of the glycoproteins in which hydroxyl groups of the carbohydrate moiety have been chemically modified by acetonation, acetylation or oxidation. The freezing point depressing activity of the derivatives has been evaluated and the results suggest that the activity results from an interaction with water or ice that involves many structural features of the glycoprotein molecules.
INTRODUCTION
An increasing number of physiological functions are being attributed to glycoproteins. Most of these glycoproteins have complicated structures, and to our knowledge, in no previous case has the complete structure of a glycoprotein been determined. Relatively little information is available concerning the role played by the carbohydrate side chains in the mode of action of glycoproteins1. A group of glycoproteins has been isolated from the serum of the fish Trematomus borchgrevinki2, ~, which inhabits McMurdo Sound, Antarctica 4. The freezing point of the serum of the fish is --2.o°C, in contrast to the value of --0.5 to --0.8°C in most of the marine fishes 5, and 30% of this freezing point depression is a result of the presence of these glycoproteins*. On a molal basis they are about 200-500 times Biochim. Biophys. Acta, 263 (1972) 4o6-413
GLYCOPROTEINSFROM AN ANTARCTIC FISH
407
more effective than NaC1 in lowering the freezing point of water 3,e. Therefore, we can conclude that their interaction with water involves something other than the normal colligative properties shown by ideal solutes. These glycoproteins in contrast to most glycoproteins, have a very simple composition and a readily detectable activity. They are present in a range of molecular weights, but each is composed of the same repeating unit, -Ala-Thr-Ala -7, in which the threonine residue bears a disaccharide that was shown by Smith degradation s to be D-galactopyranosyl-2-acetamido-2-deoxy-D-galactopyranose. Two uncertainties about the structure of the carbohydrate moieties remained: (I) the nature of the configuration at each of the anomeric carbons, and (2) whether the galactose to N-acetylgalactosamine linkage is 1-->3 or 1-+ 4. Previous studiesS, 9, have shown that the carbohydrate moieties of these glycoproteins are involved in tile freezing point depressing activity. Borate ions, which are known to complex readily with cis-hydroxyl groups on adjacent carbon atoms 1°, destroy the freezing point depressing activity of these glycoproteins9. Acetylation of only 30% of the hydroxyl groups has also been shown to destroy their activity s, as does treatment with sodium periodate s, or removal of the terminal sugar residue of the oligosaccharide side chains by Smith degradation s. The structure of the carbohydrate moieties has been determined in detail, thereby completing the structure determination of this group of glycoproteins. Knowledge of the complete structure of the carbohydrate moieties has permitted specific modification of hydroxyl groups of the carbohydrate moieties in order to determine the role they play in imparting the unique degree of freezing point depressing activity to this group of glycoproteins. MATERIALS AND METHODS The freezing point depressing glycoproteins (I) were isolated from the blood serum of T. borchgrevinki according to the method of De Vries 2 and De Vries et al. 3. All studies were carried out on a sample of the glycoproteins containing the natural mixture of three components with molecular weights of io 500, 17 ooo and 21 500. A sample of acetylated glycoproteins for NMR analysis was prepared according to the method of Komatsu et al. s. The glycodipeptide (II) was prepared from subtilisindigested glycoproteins according to the method of De Vries et al. 7. a-D-Galactosidase 11 (from fig latex) and/~-D-galactosidase (from jack bean meal) were supplied by Dr Y. T. Li of Tulane University, Covington, La. Galactose oxidase (Galactostat Kit) was purchased from Worthington Biochemical Co. Methylated bovine serum albumin and lactose were obtained from Sigma, deuterated solvents from Mallinckrodt, and lactnlose from Nutritional Biochemical Co. Lactal and/~-D-galactopyranosyl-(I-+4)a-methyl-D-mannopyranoside were synthesized by the method of Haworth et al. TM. NMR spectra were obtained on either a Varian Associates HR-22o or a J E O L JNM PSIoo instrument in deuterated solvents. Incubation of glycoproteins with a- and'~-galactosidases A sample of glycoproteins (3 rag) was dissolved in 15o ~ul of 0.05 M sodium acetate buffer (pH 4-5), and to this solution 3o/~1 of enzyme (I unit enzyme per IO #1 ; one unit of enzyme is defined as that amount which hydrolyzes I #mole of p-nitroBiochim. Biophys. Acta, 263 (1972) 4o6-413
4 °8
W.T. SHIER et al.
phenyl-a-I)-galactoside or p-nitrophenyl-fl-D-galactoside per min at 25°C) were added. The incubation was carried out at 37°C for 72 h (Y. T. Li, personal communication). After incubation, 20 pl of the reaction mixture was used directly for paper chromatography according to the method of Trevelyan et al. TM. Galactose was used as a standard.
I sopropylidene glycoproteins (III) Glycoproteins (5o rag) were dissolved in a mixture of dimethylformamide (2 ml) and 2,2-dimethoxypropane (I ml). A few crystals of p-toluene sulfonic acid were added as a catalyst and the mixture was stirred at room temperature for 24 h (ref. 14). Sufficient K2CO 3 was added to neutralize the p-toluene sulfonic acid, the salt was removed from the solution by dialysis, and the solution was lyophilized. The NMR spectrum of I I I in ~H20 was similar to that of native glycoproteins, but contained additional absorptions at 6 1.32 p p m to 6 1.54 p p m overlapping with the C-CH 3 absorptions due to alanyl and threonyl residues. Integration of the complex absorption indicated that one isopropylidene group was present for each acetamido group (6 2.00 ppm).
Acetylated isopropylidene glycoproteins (IV) Compound I I I was acetylated according to the method of Komatsu et al. s. The NMR spectrum in E~Hlchloroform contained absorptions at ~ 1.44 to 1.54 p p m (15 H, of which 9 H is due to alanyl and threonyl methyl groups) indicating the retention of a single isopropylidene group, and absorptions at ~ 2.04 p p m (3 H, acetamido group) and at 6 2.12 to 2.25 p p m (12 H, four acetoxyl groups).
Selectively acetylated glycoproteins (V) Compound V was prepared from Compound IV by acid hydrolysis according to the method given below. The NMR spectrum in I2H61dimethylsulfoxide contained no isopropylidene absorption, but contained a complex peak centering at 6 2.Ol p p m (12 H, acetoxyl) and at 6 1.88 ppm (3 H, acetamido).
Aldehydoglycoproteins (VI) Native glycoproteins were oxidized with galactose oxidase according to the method of De Vries 6. Monitoring the reaction indicated oxidation of 1.6 hydroxyl groups per disaccharide residue. The enzymes were separated from the reaction mixture using a DEAE-cellulose column in 0.0025 M Tris buffer (pH 9.6). The aldehydroglycoproteins fraction was collected, dialyzed and lyophilized. Although the NMR spectrum did not show any aldehyde absorptions in the expected region, the product of the reaction reacted like an aldehyde when oxidized with bromine water, and it gave acetylated derivatives (vide infra) that corresponded to oxidation of both sugar residues of the disaccharide moiety. In addition, it migrated at the same rate as native glycoproteins on acrylamide-gel electrophoresis carried out according to the method of De Vries et al. ~, and it did not form an insoluble complex with methylated bovine serum albumin, indicating that no over oxidation to carboxyl groups had occurred. Biochim. Biophys. Acta, 263 (1972) 4o6-413
GLYCOPROTEINS FROM AN ANTARCTIC FISH
409
Carboxyglycoproteins (VII) Compound VI (4 ° mg) was dissolved in water (2 ml), and CaCO 3 (o.5 g) and bromine (o.o15 ml) was added. The mixture was allowed to stand with occasional shaking for 2 h. The excess Br 2 was destroyed with sodium thiosulfate solution and the mixture was acidified with I.O M HC1 until the excess CaCO 3 dissolved. The mixture was dialyzed and lyophilized to yield 4 ° mg of Compound V I I that forms an insoluble complex with methylated bovine serum albumin and migrates much faster than native glycoproteins on acrylamide-gel electrophoresis.
I sopropylidene aldehydoglycoproteins (VIII) Aldehydoglycoproteins were converted to the isopropylidene derivatives by the method used for the preparation of Compound I I I . The NMR spectrum contained absorptions in the range d 1.32 to 1.54 p p m that integrated for one isopropylidene group per disaccharide residue.
A cetylated isopropylidene aldehydoglycoproteins (IX) Compound V I I I was acetylated according to the method of Komatsu et al.s. The NMR spectrum contained absorptions at ~ 1.22 to 1.53 p p m (complex peak, 15 H, indicating one isopropylidene group per disaccharide residue), ~ 2.00 p p m (3 H, acetamido) and d 2.15 p p m (complex peak, 6 H, acetoxyl).
Selectively acetylated aldehydoglycoproteins ( X ) Compound I X was subjected to mild acid hydrolysis as described below to give Compound X. The NMR spectrum contained no isopropylidene absorptions, but 2.04 p p m (3 H, acetamido), ~ 2.12 p p m (3 H, acetoxyl) and ~ 2.18 p p m (3 H, acetoxyl).
Acid hydrolysis of isopropylidene derivatives Isopropylidene derivatives were mixed with HC1 to give a final concentration of o.oi M, and the mixture was incubated at 95°C for 20 min. The reaction mixture was dialyzed and lyophilized. The NMR spectrum of the products showed no isopropylidene absorptions.
Deacetylation Removal of acetyl groups was carried out in a hydroxylamine solution according to the method of Grossberg and Pressman 15.
Determination of freezing points The freezing point measurements were made with a Fiske Osmometer calibrated with standard NaC1 solutions 3. Since the acetyl and isopropylidene derivatives were not soluble in water, the freezing points of their solutions were determined in i M aqueous dimethylformamide, and compared with the freezing point of solutions of native glycoproteins in the same solvent. RESULTS
Structure of the carbohydrate moiety The NMR spectrum of the native glycoproteins was unsatisfactory for the assignment of the stereochemistry about the two anomeric carbon atoms, since it Biochim. Biophys. Acta, 263 (1972) 4o6-413
41o
w.T. SHIER et al.
H0
~.~ fHzOH HO.v O
/'~ Hb H
CHzOH AcHN~ Ha
O(~HCH, ?H, HtNCHGONHCHCOaH
Fig. I. T h e s t r u c t u r e of t h e glycodipeptide (II) isolated from t h e subtilisin digested glycoproteins. Ha a n d Hb are t h e a n o m e r i c p r o t o n s of t h e G a l N A c a n d Gal residues, respectively. Ac is t h e acetyl group.
contained additional signals in the region of interest. However, the NMR spectrum of the glycodipeptide fragment, II (Fig. I), obtained by subtilisin digestion 7 of the glycoproteins, contained two sharp doublets at $ 4.86 ppm and $ 4.27 ppm, shifted downfield from the other C-H signals and in the region characteristic of anomeric protons 16. The coupling constants of J1,2 - 3 .6 and 7.2 Hz, respectively, suggest one a-linkage and one /~-linkage. Comparison of the chemical shifts of these anomeric protons with those of several reference compounds presented in Table I permits the assignment of a r-configuration to the galactose to N-acetylgalactosamine linkage 17, and hence, an a-configuration to the N-acetylgalactosamine t o threonine linkage. The assignment of a r-linkage to the galactosyl residue was substantiated by the results of the action of a- and ~-galactosidases on the glycoproteins. Only/~-galactosidase was able to release galactose from the glycoproteins. TABLE I NMR
DATA FOR
THE
ANOMERIC
Compound
PROTONS
Anomeric proton of galactosyl residue (ppm)
fl-Gal /%Gal /~-Gal fl-Gal II
(1--+4) (1-+4) (1--+4) (i-~4)
OF GALACTOSE-CONTAINING
Glc Glucal a-Man-OCH 3 Fru
4.23 4.39 4.31 4.43 4.27
DISACCHARIDES
Anomeric proton of other residue
Jz,2 (Hz)
c9 (ppm)
7.2 7 .2 6.8 6.8 7.2
4.99
4.0
-
-
-
4.64 -4.86
.[1,2 (Hz)
-
2. 4 -3.6
An analysis of the NMR spectrum of the acetylated glycoprotein permits the assignment of a I--~4 galactose to N-acetylgalactosamine linkage. In the NMR spectra of acetylated sugars the~absorptions of axial secondary acetoxyl groups can generally be resolved from those of the equatorial secondary acetoxyl groups, and also from acetamido and primary acetoxyl group absorptions in. On acetylation the C-4 hydroxyl~group of the N-acetyl-o-galactosamine residue in the native glycoproteins will give an axial secondary acetoxyl group unless that hydroxyl group is involved in the glycosidic linkage. That is, if the linkage is I---~4, one axial secondary acetoxyl group (on the I)-galactose residue) will be formed, but if the linkage is I --~3, two will be formed. In the NMR spectrum of the acetylated glycoproteins the acetyl Biochim. Biophys. Acta, 263 (1972) 4o6-413
GLYCOPROTEINS FROM AN ANTARCTIC FISH
411
absorptions were partially resolved into the following peaks: 6 1.95 ppm, 3 H, assigned to the acetamido group16; 6 2.03 ppm with a shoulder at ~ 2.08 ppm, 9 H, assigned to the equatorial secondary acetoxyl groups16; a doublet at d 2.15 ppm and 2.17 ppm, 6 H, assigned to the primary acetoxyl groups; and b 2.22 ppm, 3 H, assigned to a secondary axial acetoxyP n. The observed pattern of relative intensities (i.e. 1:3:2 :I) is consistent with a 1-+ 4 linkage, but is inconsistent with a 1-+3 linkage which would require a 1:2:2:2 pattern of relative intensities. Hence, the structure of the disaccharide-threonine moiety is fl-n-galactopyranosyl-(I-+4)-a-2-acetamidodeoxy-n-galactopyranosylthreonine (Fig. I). The freezing point depressing activities of the chemically modified glycoproteins were determined and compared with the activity of the native glycoproteins. The data are summarized in Table II. The formation of isopropylidene derivatives and acetylation did not cause any permanent disruption of the molecule since removal of these groups by mild acid hydrolysis and hydroxylamine, respectively, always regenerated the original activity. TABLE
II
FREEZING
POINT
DEPRESSING
ACTIVITY
OF CHEMICALLY
MODIFIED
GLYCOPROTEINS
R,O
[
R,
o
,,o<.Z-o,/hAo ORs Ac HN
I 0 I -~Ala--Thr--Ala--)- n
Compound
Structure**
I III IV V VI VII VIII IX X
Freezing point depressing activity*
R I = Rs = H, R z = CH,OH (R1) ~ = > C ( C H a ) 2 . R , = C H 2 O H , R a = H (R1) 2 = > C ( C H 3 ) = , R~ = C H 2 O A c , R z = Ac** R x = H , R~ = C H , O A c , R n = A c R 1 = R a = H , R~ = C H O R a = R 3H, R 2 = CO2H (R1) 2 = > C ( C H 3 ) v R~ = C H O , R 3 = H ( R , ) 2 = > C ( C H 3 ) 2, R 2 = C H O , R 3 = A c R~ : H , R~ : C H O , R~ = A c
IOO <5 <5 <5 95 <5 <5 <5 <5
" T h e f r e e z i n g p o i n t d e p r e s s i n g a c t i v i t y o f C o m p o u n d I, n a t i v e g l y c o p r o t e i n s , w a s a s s i g n e d a s i o o . T h e a c t i v i t i e s o f o t h e r c o m p o u n d s w e r e e x p r e s s e d r e l a t i v e t o C o m p o u n d I. "* A c is t h e a c e t v l g r o u p .
DISCUSSION
The structure determined for the disaccharide moieties of the freezing point depressing glycoproteins of T. borchgrevinki shows considerable homology with the oligosaccharide moieties of other glycoproteins. As in all other adequately studied Biochim. Biophys. Ac/a,
263 (1972) 4 o 6 - 4 1 3
412
w.T. SHIER et al.
glycoproteins that contain an N-acetylgalactosamine-threonine linkage, the linkage is of the a-D-anomeric configuration is. The/~-D-galactopyranosyl-(i-+4)-2-acetamido2-deoxygalactopyranose structure that constitutes the disaccharide moieties of freezing point depressing glycoproteins also forms part of a trisaccharide isolated from porcine submaxillary mucin by Katzman and Eylar 19. The disaccharide may also form part of the oligosaccharide of M blood-group substance s°. It has been reported previouslyS,9 that the carbohydrate moieties are essential for the activity of the freezing point depressing glycoproteins. The results presented in this report support and extend this conclusion. The observation that most of the modified glycoproteins are inactive implies that most of the functional groups in the disaccharide moieties are involved in the interaction with water. Several plausible mechanisms by which the glycoproteins may depress the freezing point of aqueous solutions have been put forward 9. The results presented in this report do not permit one to distinguish between models in which depression of the freezing point results from the structuring of water by hydrogen bonding or by inhibition of ice crystal growth through surface adsorption. If a hypothesis featuring water structuring by means of hydrogen bonding is correct, it would appear that the freezing point depressing activity results from a rather specific structuring of water, rather than merely the amount of water ordered, because the carboxyl groups of the inactive carboxyglycoproteins would be expected to order more water in their hydration spheres than aldehyde or hydroxymethyl groups of the active aldehydo- and native glycoproteins, respectively, could order by hydrogen bonding. However, it is difficult to reconcile a water structuring hypothesis with the thermal hysteresis observed in the freezing and melting behavior of solutions of the glycoproteins9. The thermal hysteresis is better explained by the model where the glycoproteins bind to the surface of crystals preventing ice formation and hence lowering of the freezing point. Experiments are in progress to determine what other features of the structure of these glycoproteins are responsible for their freezing point depressing activity. ACKNOWLEDGMENTS
This work was supported in part by National Science Foundation Grant GV27327, Dernham Junior Fellowship J-I58 from the American Cancer Society, California Division (to W.T.S.), and National Institutes of Health Postdoctoral Fellowship I FO2HE5o4o5-oI (to Y.L.). We thank Dr John Wright of the University of California and Mr R. Kaiser of the Salk Institute for obtaining the NMR spectra, and Dr Y. T. Li of Tulane University for kindly supplying a- and fi-galactosidases. REFERENCES
R. G. Spiro, Annu. Rev. Biochem., 39 (197 o) 599. A. L. DeVries, P h . D . Thesis, Stanford University, 1968. A. L. DeVries, S. K. K o l n a t s u and R. E. Feeney, J. Biol. Chem., 245 (197 o) 29Ol. A. L. DeVries and D. E. Wohlschlag, Science, 163 (1969) lO73. V. S. Black, Univ. Toronto Biol. Ser., 59 (1951) 53. A. L. DeVries, in W. H. H o a r and D. J. Randall, Fish Physiol., Vol. 6, Academic Press, New York, I971, p. 157. 7 A. L. DeVries, J. Vanderheede and R. E. Feeney, J. Biol. Chem., 246 (1971) 305 . 8 S. K. K o m a t s u , A. L. DeVries and R. E. Feeney, J. Biol. Chem., 245 (197o) 29o9.
I 2 3 4 5 6
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9 A. L. DeVries, Science, 172 (1971) 1151. i o C. A. Zittle, Adv. Enzymol., 12 (1952) 439i i S. I. Hakomori, B. Siddiqui, T.-Y. Li a n d S.-C. Li and C. G. Hellerqvist, J. Biol. Chem., 246 (1971 ) 2271. 12 W. N. H a w o r t h , E. L. Hirst, M. M. T. P l a n t and R. J. W. Reynolds, J. Chem. Soc., (193 o) 2644. 13 W. E. Trevelyan, D. P. P r o c t e r a n d J. S. Harrison, Science, 166 (195 o) 444. 14 M. E. E v a n s , F. W. P a r r i s h and L. Long, Jr., Carbohydr. Res., 3 (1967) 453. 15 A. L. Grossberg and D. P r e s s m a n , Biochemistry, 2 (1963) 90. 16 L. D. Hall, Advan. Carbohydr. Chem., 19 (1964) 51. 17 J. M. VAN DER VEEN, J. Org. Chem., 28 (1963) 364 . 18 R. D. Marshall a n d A. Neuberger, Adv. Carbohydr. Chem. Biochem., 25 (197 o) 4o7 • 19 R. L. K a t z m a n and E. H. Eylar, Biochem. Biophys. Res. Commun., 23 (1966) 769 . 20 D. B. T h o m a s and R. J. Winzler, Biochem. Biophys. Res. Commun., 35 (1969) 811.
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