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Properties of carbohydrate-stripped thyroglobulin
642
BIOCHIMICA ET BIOPHYSICAACTA
BBA 35773 P R O P E R T I E S OF C A R B O H Y D R A T E - S T R I P P E D T H Y R O G L O B U L I N I. PREPARATION AND PHYSICOCHEMICAL P R O P E R T I E S OF DESIALIZED T H Y R O G L O B U L I N
OSAMU T A R U T A N I * AND S I D N E Y S H U L M A N
Department of 3lwrob,ology, New York Medical College, New York, N . Y . lOO29 (U.S.A.)
SUMMARY
The influence of sialic acid on the conformation of thyroglobulin was investigated by comparing largely desialized thyroglobulin with native (intact) thyroglobulin. There was no significant difference in the ultraviolet absorption spectrum nor in the dissociation into subunits produced by treatment with sodium dodecyl sulfate, at least, around neutral pH. The results from sedimentation studies show that both protein preparations are fairly compact molecules. These data indicate that no appreciable gross conformational changes take place on removal of the sialic acid residues from thyroglobulin. However, there were slight differences in electrophoretic mobility between native and desialized thyroglobulin, and there was partial aggregation of desialized thyroglobulin at pH 5.6. Furthermore, the contribution of sialic acid residues to the acidic properties of thyroglobulin was shown by a change from a single boundary to two boundaries, and by the rise in the isoelectric point from 4.49 to the values of 4.55 and 4.66 for these two main peaks that were detected for desialized human thyroglobulin, as shown by isoelectric focusing.
INTRODUCTION
In recent years, the importance of carbohydrate residues has been demonstrated with regard to the physicochemical, biological, and immunological aspects of various glycoproteins. In thyroglobulin, the composition and content of the carbohydrate units and their incorporation into pre-thyroglobulin or immature thyroglobulin molecules has been studied in several laboratories. However, there is still no information concerning the contribution of carbohydrate to the physicochemical or immunological properties of the native (intact) thyroglobulin molecule. Therefore, an attempt was made to prepare sialic acid-poor thyroglobulin without any risk of denaturation, in order to clarify the properties of desialized thyroglobulin. This sugar * Present address: D e p a r t m e n t of Physical Biochemistry, I n s t i t u t e of Endocrinology, G u n m a University, Maebashi, Japan.
Bioch~m. Biophys. Acta, 229 (I97 I) 642-648
CARBOHYDRATE-STRIPPED THYROGLOBULIN
643
unit, sialic acid, was chosen because it is probably located at the terminal position of the carbohydrate chains in the thyroglobulin molecule, as indicated by the work of SPIRO AND SPIRO1,~. It was also chosen to clarify the influence of sialic acid residues on the conformation of thyroglobulin, because sialic acid residues, being negatively charged groups, make a strong contribution to the surface charge of a glycoprotein. The reasons that human and ovine thyroglobulin were chosen are the following: human thyroglobulin is interesting in regard to its autoimmune and heteroimmune antigenic properties 8-5, and ovine thyroglobulin is expected to show a more striking charge alteration by desialization, because of its higher content of sialic acid, compared to that in other animal species. A preliminary report on this work has appeared 6. MATERIALS AND METHODS
Materials The thyroglobulin was prepared from normal human thyroid glands (4 glands which were removed at autopsy) and from ovine thyroid glands which were purchased from Pel-Freez Biologicals, by fractional salting-out with 1.5o to 1.8o M of (NH4),SO ~, performed at low temperature. This fraction was further purified by gel filtration (2.5 cm × 96 cm column) on 6% agarose gel (Bio-Gel A-5m from Bio-Rad Labs., Calif.). For further purification, the gel filtration product was applied to DEAEcellulose chromatography in a manner similar to that reported elsewhere ~. This thyroglobulin preparation was completely free of proteolytic activity. Measurement and removal of sialic acid The content of sialic acid in the thyroglobulin was measured directly in the incubation mixtures by the thiobarbituric acid assay of WARREN'S8 method, without neutralization, after the liberation of protein-bound sialic acid by means of 0.025 M H2SO 4 hydrolysis at 80 ° for 60 min, or by the combined action of 24 h of pronase digestion, followed by this mild acid hydrolysis, or by digestion with neuraminidase at neutral pH. Enzymatic reactions were carried out at 37 ° for various durations. The neuraminidase of Vibrio cholerae, 500 units/ml, obtained from Calbiochem did not show proteolytic activity or any other glycosidase activity on incubation with thyroglobulin, when tested by paper chromatography, after passing the reaction mixture through Dowex 5 ° (H +) and I(CO32-). There were no galactose, mannose, N-acetylglncosamine or fucose spots. Furthermore, the MORGAN-ELsoN9 method for hexosamine proved also to be negative. These additional sugar analyses were done by Dr. T. Muramatsu of the Department of Biophysics and Biochemistry of Tokyo University, Japan. Thyroglobulin and neuraminidase, in final concentrations about 5 mg/ml and 25 units/ml, respectively, were incubated in phosphate-NaC1 buffer of pH 7-5 or phosphate buffer of pH 6.5. In this paper, desialized thyroglobulin used for the study of structural properties was prepared by treatment with neuraminidase for 24 h at 37 °, except where otherwise noted. Determination of thyroglobulin concentration An extinction coefficient of lO. 4 for a 1% solution at 28o m# was used for determination of thyroglobulin concentration lo. Bzochim. Biophys. Acta, 229 (1971) 642-648
644
o . TARUTANI, S. SHULMAN
%
.# o 05
¸
i
J ;o
,~
2'0
/ / 15
Incubation (hrs)
Fig. I. Release of sialic acid plotted against time of incubation for h u m a n thyroglobulin, treated w i t h n e u r a m i n i d a s e at p H 7-5, ionic s t r e n g t h 0.2, and 37 °. The value for total release, obtained b y t r e a t m e n t with pronase plus acid, is also indicated - - ( D - - .
Measurement of isoelectrie point by isoelectrie focusing The isoelectric points of native and desialized thyroglobulin were determined by Prof. N. Ui of the Institute of Endocrinology, Gunma University, Japan, using an isoelectric focusing column of IiO ml (LKB Instruments) with a carrier of synthetic ampholyte. The isoelectric points of the ampholyte were distributed between pH 4 and 6. Experiments were carried out at 5 °, RESULTS
Removal of sialic acid from the thyroglobulin molecule Fig. I shows a typical study on the amount of sialic acid that is released, reaching a maximum after 22 h of incubation with neuraminidase at pH 7.5. Even further addition of neuraminidase after 24 h of incubation cannot release more sialic acid. Nonetheless, this value is smaller than that obtained when thyroglobulin is treated by the combined action of pronase digestion and mild acid hydrolysis. The illustrative comparisons in Table I show that this combined action of pronase and acid gives the highest value of sialic acid released, as compared to the TABLEI A M O U N T OF S l A L I C A C I D R E L E A S E D
FROM THYROGLOBULIN
Thyroglobulin
Treatment*
Siahc acid (rag/zoo mg thyroglobulin)
% Release of smlic acidfrom thyroglobulin
Human
P r o n a s e + acid Neuraminidase Acid
1.o 7 o.88 o.93
ioo 82 87
Ovine
P r o n a s e + acid Neuraminidase Acid
1.52 i .41 i .48
IOO 93 97
* E n z y m e t r e a t m e n t s included 24 h of incubation.
Bioch,m. Biophys. Acta, 229 (1971) 642-648
BY DIFFERENT
TREATMENTS
CARBOHYDRATE-STRIPPED THYROGLOBULIN
645
action of neuraminidase or acid, separately, which show about 82 ~o or 87~o, respectively, of sialic acid release in human thyroglobulin, and 93 % or 97 % in ovine thyroglobulin. Therefore, this combined action of pronase and acid is thought to cause the complete release of sialic acid from thyroglobulin and is taken to be the IOO~o value. Similar total content (1.o7 mg sialic acid for IOO mg of human thyroglobulin and about 1.5 mg for ovine thyroglobulin) were reported previously by SPIRO AND SPIRO 1 and BOUCHILLOUXet al. 11. The enzymatic action of the neuraminidase used here is very similar throughout the range of pH between 5.6 and 7.5, and the thyroglobulin remains soluble in the neutral pH region during the one or two days of incubation. When 1.5 mM or IO mM sodium dodecyl sulfate, which dissociates thyroglobulin into subunitslZ, la, is added to thyroglobulin solutions shortly before the addition of neuraminidase, the amount of sialic acid released shows a slight increase, compared to that released on treatment of native thyroglobulin, even though it seems that 5o-6o~o of thyroglobulin dissociates into its subunits under these conditions. However, this increment is only approx. 3-5 % and it should not be considered significant.
Structural properties of the desialized thyroglobulin molecule The ultraviolet absorption spectrum and sedimentation coefficient of a desialized thyroglobulin preparation are similar to those of native thyroglobulin around neutral pH. Therefore, it seems that neuraminidase treatment does not cause gross changes in the thyroglobulin molecule. However, partial aggregation occurs at acidic pH. Fig. 2 shows typical sedimentation patterns of native and desialized thyroglobulin preparations under various conditions. In native thyroglobulin (Fig. 2a), there is a single peak with the normal sedimentation coefficient of 18. 4 S at pH 5.6. In desialized thyroglobulin at the same pH (Fig. 2b), however, partial aggregation occurs, giving sedimenting material between 19 S and 2 7 S. The feature of dissociation into subunits by treatment with sodium dodecyl sulfate is quite similar at neutral pH to the results with intact thyroglobulin. At IO mM of sodium dodecyl sulfate, in the solution of phosphate-NaC1 buffer, pH 7.5
Fig. 2. Typical ultracentrifugal patterns of human thyroglobulin in both the native and desialized forms, and at several levels of p H and sodium dodecyl sulfate, a. Native protein (upper, o.2 % ; lower, o.8 %) at pH 5.6, zero concentration sodium dodecyl sulfate, b. Desialized protein (upper, o.2 %, lower, 0. 7 %) at pH 5.6, zero concentration sodium dodecyl sulfate, c. Native protein (upper, 0. 4 %) and desialized protein (lower, 0. 4 %) at pH 7.5, Io mM sodium dodecyl sulfate, d. Native protein (upper, 0.4% ) and desialized protein (lower, o.4% ) at pH 5.6, o.5 mM sodium dodecyl sulfate. Direction of sedimentation is to the right.
Bioch*m. B,ophys. Acta, 229 (1971) 642-648
646
O. TARUTANI, S. SHULMAN
and ionic strength o.I, dissociation into a subunit of S-I2 component reached 49% in intact thyroglobulin and 52% in desialized thyroglobulin after correction for the Johnston-Ogston effect, as shown in Fig. 2c. The dissociation pattern of desialized thyroglobulin in the presence of sodium dodecyl sulfate at pH 5.9 is closely similar to that of native thyroglobulin. However, the dissociation pattern at more acidic pH is quite different. Desialized thyroglobulin shows a very diffuse pattern, even at low concentration of sodium dodecyl sulfate (Fig. 2d). Therefore, at low pH, there is more lability, and dissociation occurs into components sedimenting more slowly than the I2-S subunit. Employing electrophoretic examination on cellulose acetate membrane, desialized thyroglobulin always shows less mobility to the anode than does native thyroglobulin, above pH 5. The difference of mobility is apparently larger in ovine thyroglobulin than in human thyroglobulin. The mobility of desialized thyroglobulin is about 90% of native thyroglobulin in human at pH 6.5, and about 80% in ovine around pH 5.6. This reduction in mobility would be expected after removal of strongly acidic groups, and this effect is larger in ovine thyroglobulin because its sialic acid content is larger than that of human thyroglobulin. However, from the approximate plots of electrophoretic mobility at several pH levels between 3.6 and 5.6, an isoelectric point shift upward by desialization of only o.I pH is estimated for human thyroglobulin, which preparation is 8o% stripped in sialic acid content. About 0.2 pH unit is estimated for the shift of isoelectric point for ovine (90% stripped) thyroglobulin.
l
a
I./'t6
N~ 0 I0 ,,'" 000
4
I0
20
," 30
,
40
50
Fr action number
b
.-/ 6
?
0 2O
~
0 I0 0 O0 I0
.,-;" 20
30
40
50
Frcmhon number
Fig. 3, Distribution of total protein in isoelectric focusing of human thyroglobulin. Light absorption and p H values are both plotted against e l u t i o n f r a c t i o n number, a. Native protein, b. Desialized protein. In e a c h case, a i % s o l u t i o n w a s a p p l i e d t o t h e c o l u m n a n d t h e r u n w a s m a d e at 5 °. Arrows indicate isoelectric points.
For more precise determination of the isoelectric point, isoelectric focusing was applied. As seen in Fig. 3, these data indicate that the isoelectric point of human thyroglobulin, normally 4.49, shifts upward only about o.I pH unit by desialization, and in the case of ovine thyroglobulin, the isoelectric point shifts about 0.3 pH unit, from the normal value at pH 4.5 to the value of 4.8, showing a slightly larger shift B,ochzm. Bzophys Acta, 229 (I97 t) 642-648
CARBOHYDRATE-STRIPPED THYROGLOBULIN
647
than that in the human protein. Moreover, the desialized human thyroglobulin distribution curve shows two main peaks, with values of 4.55 and 4.66; the mean value, considering the total pattern as a single distribution, was 4.6, representing a broader peak than for intact thyroglobulin (Fig. 3). This kind of micro-difference is never detected in zone electrophoresis.
DISCUSSION
As seen in Table I, neuraminidase digestion, or even simple acid treatment, cannot release lOO% of the sialic acid, but yields only an incomplete release of about 82 to 87% in human, and 93 to 97°/'0 in ovine, thyroglobulin. The I2-S subunit when treated with sodium dodecyl sulfate also shows the same limitations, in that the total sialic acid, as measured in the native thyroglobulin, is not released completely. From these data we can assume that the incomplete release is probably due to steric hindrance in the thyroglobulin molecule, i.e. a certain amount of sialic acid may be located in the interior of the thyroglobulin molecule because of its large molecular size, and neuraminidase or acid can release only the sialic acid which is located on the surface of the thyroglobulin molecule. The cleavage of polypeptide chains with pronase might well expose the hidden sialic acid, and this would lead to the more complete release of sialic acid with acid treatment. The relative amounts of sialic acid removed by treatment with neuraminidase or acid are larger in ovine than in human thyroglobulin. This result indicates that the amount of sialic acid located on the surface of the molecule is larger in ovine thyroglobulin. Concerning desialization without any risk of denaturation of a glycoprotein, most data show that there are no gross conformational changes taking place on removal of the sialic acid from fetuin14,15, and ayacid glycoprotein TM, as judged from a number of physicochemical measurements; there are merely some changes in isoelectric point and titration properties. The only striking structural change that has been reported was a difference in crystalline structure between native and desialized ceruloplasmin 17. In the present work, it is important to note that thyroglobulin also showed no appreciable change in tertiary structure through desialization around neutral pH, as judged from its ultraviolet spectrum, sedimentation velocity, and dissociation into subunits, even though desialized thyroglobulin is more labile at acidic pH. However, structural change is indicated by the isoelectric focusing examination shown in Fig. 3. The isoelectric point of thyroglobulin, which is 4-49 in human, shifts into two peaks; one at 4.55 and the other, 4.66, after the sialic acid has been removed. In ovine thyroglobulin, the shift is from 4-5 to 4.8. These shifts are much smaller than those in other glycoproteins, such as fetuin 1. and plasma acid glycoprotein~8,TM. It seems that this difference depends on the sialic acid content, since fetuin and plasma acid glycoprotein contain 8% and IO%, respectively, in contrast to only 1% in human, or 1.5°//o in ovine, thyroglobulin. Interestingly, the distribution of the isoelectric focusing patterns became broader and revealed two peaks upon desialization, though electrophoresis experiments showed only a single band at various pH levels. These results may suggest the possibility of some heterogeneity in the desialized thyroglobulin, for example, Biochtm. Btophys. Acta, 229 (t97 I) 642-648
648
o. TARUTABII, S. SHULMAN
discrete difference in the removal of sialic acid, although more detailed analysis is needed to clarify this concept *°. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Prof. N. Ui for his kind efforts and discussion in the isoelectric focusing experimentation. Thanks are also due to Dr. T. Muramatsu for the analysis of other glycosidase activities in the neuraminidase preparation. This research was aided by a grant from The John A. Hartford Foundation, Inc. REFERENCES I ]~. G. SPIRO AND M. J. SPIRO, J. Bwl. Chem., 24o (1965) 997. 2 M. J. SPIRO AND R. G. SPIRO, J. B~ol. Chem., 243 (1968) 6520. 3 I. M. ROITT, P. N. CAMPBELL AND D. DONIAOH, Biochem. J., 69 (1958) 248. 4 S. SHULMAN AND E. WITEBSKY, J. Immunol., 85 (196o) 559. 5 S. SHULMAN, Gunma Syrup. Endoerinol., 5 (1968) 117. 6 0 . TARUTANI AND S. SHULMAN, Federation Proc., 29 (197o) 645. 7 N. UI, O. TAROTANI, Y. KONDO AND H. TAMURA, Nature, 191 (1961) 1199. 8 L. WARREN,J. Biol. Chem., 234 (1959) 1971. 9 W. T. J. MORGAN AND L. A. ELSON, Biochem. J., 28 (1934) 988. IO S. SHULMAN AND E. WITEBSlCY, J. Immunol., 88 (1962) 221. i i S. BOUCHILLOUX, M, I~OLLAND, J. TORRESANI, M. ROQUES AND S. LISSITSKY, B~ochim. B1ophys. Acta, 93 (1964) 15. 12 H. EDELHOCH AND R. E. LIPPOLDT, J. B,ol. Chem., 235 (196o) 1335. 13 O. TARUTANI AND N. UI, Bzochim. Biophys. Acta, 181 (1969) 116. 14 R. G. SPIEO, J. B*ol. Chem., 235 (196o) 286o. 15 Y. OSHIEO AND E. H. EYLAR, Arch. Bzochem. B~ophys., 13o (1969) 227. 16 R. C. HUGHES AND R. W. JEANLOZ, Biochemzstry, 3 (1964) 1543. 17 A. G. MORELL, I. STERLIEB AND I. H. SCHENINBERG, Sc*ence, 166 (1969) 1293. 18 I. YAMASHINA,Acta Chem. Scand., IO (1956) 1666. 19 E. A. POPENOE AND R. M. DREW, J. Biol. Chem., 228 (1957) 673. 20 N. UI, Abstr. 6th Intern. Thyroid Conf., Wien, z97o, p. 7.
Biochim. B,ophys. Acta, 229 (I97 I) 642-648
BIOCHIMICA ET BIOPHYSICAACTA
BBA 35773 P R O P E R T I E S OF C A R B O H Y D R A T E - S T R I P P E D T H Y R O G L O B U L I N I. PREPARATION AND PHYSICOCHEMICAL P R O P E R T I E S OF DESIALIZED T H Y R O G L O B U L I N
OSAMU T A R U T A N I * AND S I D N E Y S H U L M A N
Department of 3lwrob,ology, New York Medical College, New York, N . Y . lOO29 (U.S.A.)
SUMMARY
The influence of sialic acid on the conformation of thyroglobulin was investigated by comparing largely desialized thyroglobulin with native (intact) thyroglobulin. There was no significant difference in the ultraviolet absorption spectrum nor in the dissociation into subunits produced by treatment with sodium dodecyl sulfate, at least, around neutral pH. The results from sedimentation studies show that both protein preparations are fairly compact molecules. These data indicate that no appreciable gross conformational changes take place on removal of the sialic acid residues from thyroglobulin. However, there were slight differences in electrophoretic mobility between native and desialized thyroglobulin, and there was partial aggregation of desialized thyroglobulin at pH 5.6. Furthermore, the contribution of sialic acid residues to the acidic properties of thyroglobulin was shown by a change from a single boundary to two boundaries, and by the rise in the isoelectric point from 4.49 to the values of 4.55 and 4.66 for these two main peaks that were detected for desialized human thyroglobulin, as shown by isoelectric focusing.
INTRODUCTION
In recent years, the importance of carbohydrate residues has been demonstrated with regard to the physicochemical, biological, and immunological aspects of various glycoproteins. In thyroglobulin, the composition and content of the carbohydrate units and their incorporation into pre-thyroglobulin or immature thyroglobulin molecules has been studied in several laboratories. However, there is still no information concerning the contribution of carbohydrate to the physicochemical or immunological properties of the native (intact) thyroglobulin molecule. Therefore, an attempt was made to prepare sialic acid-poor thyroglobulin without any risk of denaturation, in order to clarify the properties of desialized thyroglobulin. This sugar * Present address: D e p a r t m e n t of Physical Biochemistry, I n s t i t u t e of Endocrinology, G u n m a University, Maebashi, Japan.
Bioch~m. Biophys. Acta, 229 (I97 I) 642-648
CARBOHYDRATE-STRIPPED THYROGLOBULIN
643
unit, sialic acid, was chosen because it is probably located at the terminal position of the carbohydrate chains in the thyroglobulin molecule, as indicated by the work of SPIRO AND SPIRO1,~. It was also chosen to clarify the influence of sialic acid residues on the conformation of thyroglobulin, because sialic acid residues, being negatively charged groups, make a strong contribution to the surface charge of a glycoprotein. The reasons that human and ovine thyroglobulin were chosen are the following: human thyroglobulin is interesting in regard to its autoimmune and heteroimmune antigenic properties 8-5, and ovine thyroglobulin is expected to show a more striking charge alteration by desialization, because of its higher content of sialic acid, compared to that in other animal species. A preliminary report on this work has appeared 6. MATERIALS AND METHODS
Materials The thyroglobulin was prepared from normal human thyroid glands (4 glands which were removed at autopsy) and from ovine thyroid glands which were purchased from Pel-Freez Biologicals, by fractional salting-out with 1.5o to 1.8o M of (NH4),SO ~, performed at low temperature. This fraction was further purified by gel filtration (2.5 cm × 96 cm column) on 6% agarose gel (Bio-Gel A-5m from Bio-Rad Labs., Calif.). For further purification, the gel filtration product was applied to DEAEcellulose chromatography in a manner similar to that reported elsewhere ~. This thyroglobulin preparation was completely free of proteolytic activity. Measurement and removal of sialic acid The content of sialic acid in the thyroglobulin was measured directly in the incubation mixtures by the thiobarbituric acid assay of WARREN'S8 method, without neutralization, after the liberation of protein-bound sialic acid by means of 0.025 M H2SO 4 hydrolysis at 80 ° for 60 min, or by the combined action of 24 h of pronase digestion, followed by this mild acid hydrolysis, or by digestion with neuraminidase at neutral pH. Enzymatic reactions were carried out at 37 ° for various durations. The neuraminidase of Vibrio cholerae, 500 units/ml, obtained from Calbiochem did not show proteolytic activity or any other glycosidase activity on incubation with thyroglobulin, when tested by paper chromatography, after passing the reaction mixture through Dowex 5 ° (H +) and I(CO32-). There were no galactose, mannose, N-acetylglncosamine or fucose spots. Furthermore, the MORGAN-ELsoN9 method for hexosamine proved also to be negative. These additional sugar analyses were done by Dr. T. Muramatsu of the Department of Biophysics and Biochemistry of Tokyo University, Japan. Thyroglobulin and neuraminidase, in final concentrations about 5 mg/ml and 25 units/ml, respectively, were incubated in phosphate-NaC1 buffer of pH 7-5 or phosphate buffer of pH 6.5. In this paper, desialized thyroglobulin used for the study of structural properties was prepared by treatment with neuraminidase for 24 h at 37 °, except where otherwise noted. Determination of thyroglobulin concentration An extinction coefficient of lO. 4 for a 1% solution at 28o m# was used for determination of thyroglobulin concentration lo. Bzochim. Biophys. Acta, 229 (1971) 642-648
644
o . TARUTANI, S. SHULMAN
%
.# o 05
¸
i
J ;o
,~
2'0
/ / 15
Incubation (hrs)
Fig. I. Release of sialic acid plotted against time of incubation for h u m a n thyroglobulin, treated w i t h n e u r a m i n i d a s e at p H 7-5, ionic s t r e n g t h 0.2, and 37 °. The value for total release, obtained b y t r e a t m e n t with pronase plus acid, is also indicated - - ( D - - .
Measurement of isoelectrie point by isoelectrie focusing The isoelectric points of native and desialized thyroglobulin were determined by Prof. N. Ui of the Institute of Endocrinology, Gunma University, Japan, using an isoelectric focusing column of IiO ml (LKB Instruments) with a carrier of synthetic ampholyte. The isoelectric points of the ampholyte were distributed between pH 4 and 6. Experiments were carried out at 5 °, RESULTS
Removal of sialic acid from the thyroglobulin molecule Fig. I shows a typical study on the amount of sialic acid that is released, reaching a maximum after 22 h of incubation with neuraminidase at pH 7.5. Even further addition of neuraminidase after 24 h of incubation cannot release more sialic acid. Nonetheless, this value is smaller than that obtained when thyroglobulin is treated by the combined action of pronase digestion and mild acid hydrolysis. The illustrative comparisons in Table I show that this combined action of pronase and acid gives the highest value of sialic acid released, as compared to the TABLEI A M O U N T OF S l A L I C A C I D R E L E A S E D
FROM THYROGLOBULIN
Thyroglobulin
Treatment*
Siahc acid (rag/zoo mg thyroglobulin)
% Release of smlic acidfrom thyroglobulin
Human
P r o n a s e + acid Neuraminidase Acid
1.o 7 o.88 o.93
ioo 82 87
Ovine
P r o n a s e + acid Neuraminidase Acid
1.52 i .41 i .48
IOO 93 97
* E n z y m e t r e a t m e n t s included 24 h of incubation.
Bioch,m. Biophys. Acta, 229 (1971) 642-648
BY DIFFERENT
TREATMENTS
CARBOHYDRATE-STRIPPED THYROGLOBULIN
645
action of neuraminidase or acid, separately, which show about 82 ~o or 87~o, respectively, of sialic acid release in human thyroglobulin, and 93 % or 97 % in ovine thyroglobulin. Therefore, this combined action of pronase and acid is thought to cause the complete release of sialic acid from thyroglobulin and is taken to be the IOO~o value. Similar total content (1.o7 mg sialic acid for IOO mg of human thyroglobulin and about 1.5 mg for ovine thyroglobulin) were reported previously by SPIRO AND SPIRO 1 and BOUCHILLOUXet al. 11. The enzymatic action of the neuraminidase used here is very similar throughout the range of pH between 5.6 and 7.5, and the thyroglobulin remains soluble in the neutral pH region during the one or two days of incubation. When 1.5 mM or IO mM sodium dodecyl sulfate, which dissociates thyroglobulin into subunitslZ, la, is added to thyroglobulin solutions shortly before the addition of neuraminidase, the amount of sialic acid released shows a slight increase, compared to that released on treatment of native thyroglobulin, even though it seems that 5o-6o~o of thyroglobulin dissociates into its subunits under these conditions. However, this increment is only approx. 3-5 % and it should not be considered significant.
Structural properties of the desialized thyroglobulin molecule The ultraviolet absorption spectrum and sedimentation coefficient of a desialized thyroglobulin preparation are similar to those of native thyroglobulin around neutral pH. Therefore, it seems that neuraminidase treatment does not cause gross changes in the thyroglobulin molecule. However, partial aggregation occurs at acidic pH. Fig. 2 shows typical sedimentation patterns of native and desialized thyroglobulin preparations under various conditions. In native thyroglobulin (Fig. 2a), there is a single peak with the normal sedimentation coefficient of 18. 4 S at pH 5.6. In desialized thyroglobulin at the same pH (Fig. 2b), however, partial aggregation occurs, giving sedimenting material between 19 S and 2 7 S. The feature of dissociation into subunits by treatment with sodium dodecyl sulfate is quite similar at neutral pH to the results with intact thyroglobulin. At IO mM of sodium dodecyl sulfate, in the solution of phosphate-NaC1 buffer, pH 7.5
Fig. 2. Typical ultracentrifugal patterns of human thyroglobulin in both the native and desialized forms, and at several levels of p H and sodium dodecyl sulfate, a. Native protein (upper, o.2 % ; lower, o.8 %) at pH 5.6, zero concentration sodium dodecyl sulfate, b. Desialized protein (upper, o.2 %, lower, 0. 7 %) at pH 5.6, zero concentration sodium dodecyl sulfate, c. Native protein (upper, 0. 4 %) and desialized protein (lower, 0. 4 %) at pH 7.5, Io mM sodium dodecyl sulfate, d. Native protein (upper, 0.4% ) and desialized protein (lower, o.4% ) at pH 5.6, o.5 mM sodium dodecyl sulfate. Direction of sedimentation is to the right.
Bioch*m. B,ophys. Acta, 229 (1971) 642-648
646
O. TARUTANI, S. SHULMAN
and ionic strength o.I, dissociation into a subunit of S-I2 component reached 49% in intact thyroglobulin and 52% in desialized thyroglobulin after correction for the Johnston-Ogston effect, as shown in Fig. 2c. The dissociation pattern of desialized thyroglobulin in the presence of sodium dodecyl sulfate at pH 5.9 is closely similar to that of native thyroglobulin. However, the dissociation pattern at more acidic pH is quite different. Desialized thyroglobulin shows a very diffuse pattern, even at low concentration of sodium dodecyl sulfate (Fig. 2d). Therefore, at low pH, there is more lability, and dissociation occurs into components sedimenting more slowly than the I2-S subunit. Employing electrophoretic examination on cellulose acetate membrane, desialized thyroglobulin always shows less mobility to the anode than does native thyroglobulin, above pH 5. The difference of mobility is apparently larger in ovine thyroglobulin than in human thyroglobulin. The mobility of desialized thyroglobulin is about 90% of native thyroglobulin in human at pH 6.5, and about 80% in ovine around pH 5.6. This reduction in mobility would be expected after removal of strongly acidic groups, and this effect is larger in ovine thyroglobulin because its sialic acid content is larger than that of human thyroglobulin. However, from the approximate plots of electrophoretic mobility at several pH levels between 3.6 and 5.6, an isoelectric point shift upward by desialization of only o.I pH is estimated for human thyroglobulin, which preparation is 8o% stripped in sialic acid content. About 0.2 pH unit is estimated for the shift of isoelectric point for ovine (90% stripped) thyroglobulin.
l
a
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4
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20
," 30
,
40
50
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b
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~
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30
40
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Fig. 3, Distribution of total protein in isoelectric focusing of human thyroglobulin. Light absorption and p H values are both plotted against e l u t i o n f r a c t i o n number, a. Native protein, b. Desialized protein. In e a c h case, a i % s o l u t i o n w a s a p p l i e d t o t h e c o l u m n a n d t h e r u n w a s m a d e at 5 °. Arrows indicate isoelectric points.
For more precise determination of the isoelectric point, isoelectric focusing was applied. As seen in Fig. 3, these data indicate that the isoelectric point of human thyroglobulin, normally 4.49, shifts upward only about o.I pH unit by desialization, and in the case of ovine thyroglobulin, the isoelectric point shifts about 0.3 pH unit, from the normal value at pH 4.5 to the value of 4.8, showing a slightly larger shift B,ochzm. Bzophys Acta, 229 (I97 t) 642-648
CARBOHYDRATE-STRIPPED THYROGLOBULIN
647
than that in the human protein. Moreover, the desialized human thyroglobulin distribution curve shows two main peaks, with values of 4.55 and 4.66; the mean value, considering the total pattern as a single distribution, was 4.6, representing a broader peak than for intact thyroglobulin (Fig. 3). This kind of micro-difference is never detected in zone electrophoresis.
DISCUSSION
As seen in Table I, neuraminidase digestion, or even simple acid treatment, cannot release lOO% of the sialic acid, but yields only an incomplete release of about 82 to 87% in human, and 93 to 97°/'0 in ovine, thyroglobulin. The I2-S subunit when treated with sodium dodecyl sulfate also shows the same limitations, in that the total sialic acid, as measured in the native thyroglobulin, is not released completely. From these data we can assume that the incomplete release is probably due to steric hindrance in the thyroglobulin molecule, i.e. a certain amount of sialic acid may be located in the interior of the thyroglobulin molecule because of its large molecular size, and neuraminidase or acid can release only the sialic acid which is located on the surface of the thyroglobulin molecule. The cleavage of polypeptide chains with pronase might well expose the hidden sialic acid, and this would lead to the more complete release of sialic acid with acid treatment. The relative amounts of sialic acid removed by treatment with neuraminidase or acid are larger in ovine than in human thyroglobulin. This result indicates that the amount of sialic acid located on the surface of the molecule is larger in ovine thyroglobulin. Concerning desialization without any risk of denaturation of a glycoprotein, most data show that there are no gross conformational changes taking place on removal of the sialic acid from fetuin14,15, and ayacid glycoprotein TM, as judged from a number of physicochemical measurements; there are merely some changes in isoelectric point and titration properties. The only striking structural change that has been reported was a difference in crystalline structure between native and desialized ceruloplasmin 17. In the present work, it is important to note that thyroglobulin also showed no appreciable change in tertiary structure through desialization around neutral pH, as judged from its ultraviolet spectrum, sedimentation velocity, and dissociation into subunits, even though desialized thyroglobulin is more labile at acidic pH. However, structural change is indicated by the isoelectric focusing examination shown in Fig. 3. The isoelectric point of thyroglobulin, which is 4-49 in human, shifts into two peaks; one at 4.55 and the other, 4.66, after the sialic acid has been removed. In ovine thyroglobulin, the shift is from 4-5 to 4.8. These shifts are much smaller than those in other glycoproteins, such as fetuin 1. and plasma acid glycoprotein~8,TM. It seems that this difference depends on the sialic acid content, since fetuin and plasma acid glycoprotein contain 8% and IO%, respectively, in contrast to only 1% in human, or 1.5°//o in ovine, thyroglobulin. Interestingly, the distribution of the isoelectric focusing patterns became broader and revealed two peaks upon desialization, though electrophoresis experiments showed only a single band at various pH levels. These results may suggest the possibility of some heterogeneity in the desialized thyroglobulin, for example, Biochtm. Btophys. Acta, 229 (t97 I) 642-648
648
o. TARUTABII, S. SHULMAN
discrete difference in the removal of sialic acid, although more detailed analysis is needed to clarify this concept *°. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Prof. N. Ui for his kind efforts and discussion in the isoelectric focusing experimentation. Thanks are also due to Dr. T. Muramatsu for the analysis of other glycosidase activities in the neuraminidase preparation. This research was aided by a grant from The John A. Hartford Foundation, Inc. REFERENCES I ]~. G. SPIRO AND M. J. SPIRO, J. Bwl. Chem., 24o (1965) 997. 2 M. J. SPIRO AND R. G. SPIRO, J. B~ol. Chem., 243 (1968) 6520. 3 I. M. ROITT, P. N. CAMPBELL AND D. DONIAOH, Biochem. J., 69 (1958) 248. 4 S. SHULMAN AND E. WITEBSKY, J. Immunol., 85 (196o) 559. 5 S. SHULMAN, Gunma Syrup. Endoerinol., 5 (1968) 117. 6 0 . TARUTANI AND S. SHULMAN, Federation Proc., 29 (197o) 645. 7 N. UI, O. TAROTANI, Y. KONDO AND H. TAMURA, Nature, 191 (1961) 1199. 8 L. WARREN,J. Biol. Chem., 234 (1959) 1971. 9 W. T. J. MORGAN AND L. A. ELSON, Biochem. J., 28 (1934) 988. IO S. SHULMAN AND E. WITEBSlCY, J. Immunol., 88 (1962) 221. i i S. BOUCHILLOUX, M, I~OLLAND, J. TORRESANI, M. ROQUES AND S. LISSITSKY, B~ochim. B1ophys. Acta, 93 (1964) 15. 12 H. EDELHOCH AND R. E. LIPPOLDT, J. B,ol. Chem., 235 (196o) 1335. 13 O. TARUTANI AND N. UI, Bzochim. Biophys. Acta, 181 (1969) 116. 14 R. G. SPIEO, J. B*ol. Chem., 235 (196o) 286o. 15 Y. OSHIEO AND E. H. EYLAR, Arch. Bzochem. B~ophys., 13o (1969) 227. 16 R. C. HUGHES AND R. W. JEANLOZ, Biochemzstry, 3 (1964) 1543. 17 A. G. MORELL, I. STERLIEB AND I. H. SCHENINBERG, Sc*ence, 166 (1969) 1293. 18 I. YAMASHINA,Acta Chem. Scand., IO (1956) 1666. 19 E. A. POPENOE AND R. M. DREW, J. Biol. Chem., 228 (1957) 673. 20 N. UI, Abstr. 6th Intern. Thyroid Conf., Wien, z97o, p. 7.
Biochim. B,ophys. Acta, 229 (I97 I) 642-648