Purification and properties of blood group A-active glycoprotein from oyster viscera

426

Biochimica et Biophysica Acta, 451 (1976) 426--435 Q Elsevier/North-Holland Biomedical Press

BBA 28101 PURIFICATION AND PROPERTIES OF BLOOD GROUP A-ACTIVE GLYCOPROTEIN FROM OYSTER VISCERA

AKIRA OGAMO, TADASHI OGASHIWAand KINZO NAGASAWA School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108 (Japan)

(Received May 3rd, 1976)

Summary A blood group A active substance was isolated from an acetone-dried powder o f oyster viscera by extraction with 0.1 M NaC1 after heating a homogenate with extraction medium, in boiling water. After the removal of the acidic fraction with cetylpyridinium chloride, the separated neutral fraction was digested successively with a-amylase and amyloglucosidase to remove glycogen. The blood group A-active portion was eluted from a Sepharose 4B column and purified by DEAE-Sephadex column chromatography. The purified active substance was homogeneous by polyacrylamide gel electrophoresis, and its molecular weight was estimated as 100 000 by sedimentation equilibrium. The sugar content of the purified active substance, expressed in percentage of dry weight, was galactosamine, 16.6; galactose, 12.8; fucose, 9.9; glucosamine, 4.6; and glucose, 3.3. Sialic acid was not detected. Total amino acid c o n t e n t was 23.0% and the main constituents were threonine, proline and serine. The ORD spectrum indicated that the hexosamines were N-acetylated. Absence of glycolipid was confirmed by the analysis of fatty acid and sphingosine base. This active substance had a strong blood group A activity (0.04 pg/ml) but neither B nor H activity; it interacted with lima bean lectin but not with concanavalin A.

Introduction Since Landsteiner [1] discovered the ABO blood group system, many investigators have made efforts to purify the ABO group active substances from various sources. Although it is known that these substances are distributed widely in the biological world [2], there have been few reports on the active substances from invertebrates. The glycolipid fractions from certain shellfish (Corbicula sandal) were found to show strong inhibition of hemagglutination

427 induced with eel serum [3]. The extracts obtained from lyophilized organs of pond mussels (Anodota) were found to behave serologically like blood group H substance [4]. During an investigation of the acidic polysaccharide fraction from oyster viscera, we observed the presence of a blood group A activity in this fraction [5], and a further examination of the distribution of the activity revealed that the neutral polysaccharide fraction was highly active. Since the finding of the blood group A-active substance in marine molluscs was unusual, a detailed investigation was carried out. The present report describes the purification, and chemical and serological properties of blood group A-active glycoprotein from oyster viscera. Materials and Methods

Materials a-Amylase (a-l,4-glucan 4-glucanohydrolase, EC 3.2.1.1) from Bacillus subtilis {liquefying type), amyloglucosidase (a-l,4-a-l,6-glucan glucohydrolase, EC 3.2.1.33) from Rhizopus niveus (Pure grade) and concanavalin A were purchased from Seikagaku Kogyo Co., Tokyo. Pronase was a product of Kaken Kagaku Co., Tokyo. Oyster glycogen was obtained from acetone
428 Osborn [13]. Carbohydrate, protein and acidic substance were, respectively, stained by the periodic acid-Schiff reagent [14,15], Coomassie brilliant blue, and Alcian blue.

Other physical methods The ORD spectrum was measured on 0.5% (w/v) of a sample solution in 0.1 M NaC1 by a Jasco ORD-CD spectropolarimeter J-20. Molecular weight was measured by sedimentation equilibrium with a Spinco model E ultracentrifuge, according to the short-column method of Yphantis [16]. Concentrations of sample in 0.1 M NaCl were 0.2, 0.3 and 0.5%.

Serological methods Human erythrocytes were washed free of plasma and suspended in 0.9% NaC1 to 0.1% (v/v). Anti-A human and anti-B human serum were purchased from Midori Jfiji Co., Osaka. The extract of Ulex europeus with 0.9% NaCl was used as anti-H reagent. Each anti-blood group active substance was diluted with 0.9% NaC1 to four hemagglutinin doses after the assay of the potency of each. Blood group activity was measured by the hemagglutination-inhibition test according to the procedure described previously [5]. The activity was expressed as the minimum concentration (pg/ml) of total incubation medium inhibiting hemagglutination. Hemagglutination activity was estimated by the settling patterns after incubation of each diluted sample solution with the corresponding erythrocyte suspension at 37°C for 1 h. Interaction of purified blood group substances with concanavalin A was examined by the quantitative precipitation method [17].

Purification of blood group A-active substance Preparation of neutral fraction. Freshly shucked (de-shelled) oysters (Crastrea gigas) (12.8 kg) were obtained from an oyster farm in Hiroshima Prefecture of Japan. The viscera were homogenized and treated three times with 10 vols. of cold acetone {--10°C) to give an acetone
429 buffer (pH 5.6) and digested with 50 mg of a-amylase at 40°C for 17 h, in the presence of a few drops of toluene in the dialysis tube (Visking cellulose tubing, C-65), which was dipped in 20 1 of 0.02 M sodium acetate buffer (pH 5.6). The digested mixture was concentrated to 150 ml by ultrafiltration (membrane, HFA-300, Abcor Co., U.S.A.), adjusted to pH 4.6 with acetic acid, and incubated with 30 mg of amyloglucosidase at 40°C for 17 h in the presence of toluene. The digested mixture was adjusted to pH 6.5 with solid sodium acetate and heated at 80--90 ° C for 20 min. After dialysis, the small a m o u n t of precipitate formed was removed by centrifugation, and the supernatant was concentrated to 25 ml by ultrafiltration, then dialyzed, mixed with an equal volume of 0.2 M Tris • HC1 buffer (pH 8) containing 0.02 M CaCl2. The mixture was incubated with 5 mg of Pronase at 37°C in the presence of 3 ml of ethanol. After 24 h, 5 mg of Pronase was added and the mixture was further incubated for 24 h. The digested mixture was immersed in boiling water for 20 min and dialyzed. The precipitate formed was removed by centrifugation. All the supernatants obtained from 14 portions were combined and concentrated to 10 ml by ultrafiltration. Gel filtration on Sepharose 4B. The solution (10 ml) obtained above was put on a Sepharose 4B column (5 × 80 cm) equilibrated with 0.1 M NaC1, and eluted with 0.1 M NaC1 at a flow rate of 40 ml/h. Fractions (20 ml) were collected, and the absorbance at 231 nm and the hexose content, by the phenol/ H2SO4 method [18], were determined. The blood group A activity of each fraction was assayed by successive dilution according to the procedure of the serological method. The active fractions were combined, dialyzed and lyophilized. Ion-exchange chromatography on DEAE-Sephadex. DEAE-Sephadex A-25 was equilibrated with 0.01 M triethylamine adjusted to pH 8.5 with CO2. The blood group A-active fraction (114 mg) was dissolved in 5 ml of the same triethylamine/CO~ buffer and applied to the column (1.5 × 25 cm). The column was eluted first with 200 ml of the same buffer, and then with a linear gradient of NaC1 from 0 to 0.5 M in the same buffer at a flow rate of 20 ml/h. Fractions (10 ml) were collected and analyzed by the phenol/H~SO4 method. The fractions of each peak were combined, dialyzed and lyophilized. Results

Purification of blood group substance from oyster viscera The yield of the neutral fraction (78.7 g) was about five-times that of the acidic fraction (15.8 g) obtained from the complex formed with cetylpyridinium salt. Neither fraction showed the ability to agglutinate human erythrocytes (ABO type). On the other hand, the measurement of the blood group activity indicated that blood group A activity in the neutral fraction (52.3 pg/ ml) was more p o t e n t than that of the acidic fraction (188 ttg/ml). Neither B nor H activity was detected in either fractions. a-Amylase and amyloglucosidase were used to depolymerize the glycogen and other glucans in the neutral fraction. These enzymatic digestions were carried out in a dialysis tube surrounded by the same buffer as an inner tube to facilitate the process. The blood group A activity at each purification step is summarized in Table I. The amylase digestion followed by amyloglusidase

430 TABLE

I

PURIFICATION

OF BLOOD

GROUP

A-ACTIVE

SUBSTANCE

FROM

OYSTER

VISCERA

Purification was started from 12.8 kg of freshly shucked (de-shelled) oysters. Blood group A activity (/~g/ml) was expressed by the m i n i m u m concentration to inhibit hemagglutination. The activity of oyster g l y c o g e n was as l o w as > 5 0 0 # g / m l . P u r i f i c a t i o n step

Yield (rag)

Blood g r o u p A activity (pg/ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acetone powder 400 000 Neutral fraction 78 6 5 0 52.3 A m y l a s e digestion 3 100 2.67 A m y l o g l u e o s i d a s e digestion 1 500 0.67 Pronase digestion 1 340 0.67 Active p o r t i o n o b t a i n e d b y S e p h a r o s e 4B 114 0.17 D E A E - S e p h a d e x Fr-I 25.2 0.04 Fr-II 4.7 10.5 Fr-III 12.9 42,0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

digestion increased blood group A activity. Although the preparation obtained was not completely soluble in water and saline solution, its solubility was improved by a subsequent digestion with Pronase. The recovery of the preparation after Pronase digestion was high and the activity remained unchanged (Table I). The elution diagram of gel filtration of the glycogen-free preparation on Sepharose 4B is shown in Fig. 1. Both curves traced by the phenol/H2SO4

,,~ 400 08

06

~

o

O2

20

40 60 Tube cumber

80

100

Fig. 1. S e p h a r o s e 4B gel f i l t r a t i o n of t h e n e u t r a l f r a c t i o n a f t e r e n z y m a t i c d i g e s t i o n s b y e - a m y l a s e , a m y l o g l u e o s i d a s e a n d P r o n a s e . Fraction~ w e r e a n a l y z e d b y t h e p h e n o l / H 2 S O 4 m e t h o d (o -o) and b y m e a s u r e m e n t o f a b s o r b a n c e a t 231 n m ( t e ) . T h e b l o o d g r o u p A a c t i v i t y was e x p r e s s e d as t h e r e c i p r o c a l of t h e m a x i m u m d e g r e e o f d i l u t i o n to i n h i b i t h e m a g g l u t i n a t i o n b e t w e e n h u m a n t y p e A e r y t h o c y t e s and anti-A s e r u m . V o was d e t e r m i n e d b y the e l u t i o n p a t t e r n of blue d e x t r a n 2 0 0 0 .

431

0.8

IliaI

F r - ll"

]o.6

F r - 1TT

0.4

OA 0.2 0.2

0

10

20

30

40

Tube n u m b e r

Fig. 2. DEAE-Sephadex A-25 i o n - e x c h a n g e c h r o m a t o g r a p h y o f the b l o o d group-active p o r t i o n e l u t e d from Sepharose 4B. F r a c t i o n s w e r e analyzed b~ the phenol/H2SO 4 m e t h o d (o o). The NaCI c o n c e n t r a t i o n (o o) of e a c h f r a c t i o n w a s d e t e r m i n e d by a c o n d u c t i v i t y m e t e r .

method and by the absorbance at 231 nm showed two peaks. The first peaks at a void volume agreed with each other, b u t the second peaks were diverse in their positions. The active fractions were distributed between these two peaks. The active fractions as indicated in Fig. 1 were collected and subjected to further purification. The active portion eluted from the Sepharose 4B column was separated into three peaks by DEAE-Sephadex chromatography (Fig. 2). The first peak (Fr-I) eluted with the starting buffer had a potent activity (Table I), and two successive peaks (Fr-II and Fr-III) were obtained by elution with a gradient of NaC1 concentration. Table I shows that the ion-exchange chromatography could effectively separate a strongly active substance from weakly active ones. The yield of Fr-I from the neutral fraction was 0.03%. A comparison of the cellulose acetate electrophoretogram during several purification steps indicated that Fr-I was free from substances stained with Coomassie brilliant blue or Alcian blue (Fig. 3A). Electrophoretograms of Fr-I on polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel revealed a single band stained only by periodic acid-Schiff reagent (Fig. 3B and C). Fr-III was a weakly acidic substance with a smaller mobility than that of hyaluronic acid (Fig. 3A). Fr-II showed three sharp bands revealed by the periodic acid-Schiff reagent on polyacrylamide gel electrophoresis (Fig. 3B).

Chemical and serological properties o f purified blood group A-active substance The quantitative data of Fr-I for neutral sugars, amino sugars and amino acids are listed in Table II. Total percentage of these three groups of constituents amounted to 71.5% by weight. The ratio of neutral sugars/amino sugars/ amino acids was 1.00 : 0.78 : 0.84 by weight, or 1.00 : 0.75 : 1.33 by molar number. Galactosamine, galactose and fucose were the main constituents of sugar component, and glucosamine and glucose followed them. The ratio of galactose to total amino sugars was 0.56 by weight. The lower ratio of glucos-

432 CA)

orJgtn

1 cm

2 cm

I

I

I

I I I I I

HyQluroni¢ acid Pronose 4B - o c t i v e Fr - I Fr-IT

D D 0

D

O 0

D

e

O i I positive AB CBB p o s i t i v e PAS p o s i t i v e

(B) Omgln ~

e i

m

I

4,-J -

J ,

!> ~

I

4--

PAS positive PAS p o s i t i v e

PAS ond AB positive

e--I Glycogen

4 8 - active

Fr-I

Fr-lI

Fr-]]I

(C) Omgin

"4"®

m ~

PASand AB positive -~-- PAS positive

m I

I

®_J Ail~nnin

Fr-I

Fr- ~I

Fig. 3. E l e e t r o p h o r e t i c p a t t e r n s of t h e b l o o d g r o u p A - a c t i v e s u b s t a n c e at vm'ious p u r i f i c a t i o n steps. T h e a b b r e v i a t i o n s used in this figure are: l>fonasc, p r e p a r a t i o n a f t e r P r o n a s e d i g e s t i o n ; 4B-active, b l o o d g r o u p A-active p r e p a r a t i o n e l u t e d f r o m S e p h a r o s e 4B; Fr-[, F r - I I a n d Fz-III, f r a c t i o n s e l u t e d f r o m D E A E S e p h a d e x ; AB, A l c i a n b l u e ; CBB, C o o m a s s i e brilliant b l u e ; PAS, p e r i o d i c acid-Schiff r e a g e n t . ( A ) Cellulose a c e t a t e m e m b r a n e e l e c t r o p h o r e s i s . H y a l u r o n i c acid ( S e i k a g a k u K o g y o Co., T o k y o ) was r u n as a r e f e r e n c e s u b s t a n c e a n d was s t a i n e d with AB. (B) P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . G l y c o g e n p r e p a r e d f r o m o y s t e r viscera was r u n as a r e f e r e n c e s u b s t a n c e a n d was stained w i t h PAS. (C) S o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . S a m p l e s w e r e p r e v i o u s l y i n c u b a t e d in 0.01 M p h o s p h a t e b u f f e r ( p H 7.2) c o n t a i n i n g 1% s o d i u m d o d e e y l sulfate a n d 4 M u r e a at 1 0 0 ° C f o r 5 rain in t h e p r e s e n c e of 2m e r c a p t o e t h a n o l . Bovine s e r u m a l b u m i n ( F r a c t i o n V p o w d e r , S i g m a C h e m i c a l Co.) was r u n as a r e f e r e n c e s u b s t a n c e a n d was s t a i n e d w i t h CBB.

amine to galactosamine (0.28, by weight) was characteristic of this blood group substance. Sialic acid was not detected at all by gas chromatography and also by the thiobarbituric acid method. Hydroxy amino acids, threonine and serine, and proline which were outstanding components made up 78% of the total weight of amino acids. No trace of fatty acids or sphingosine base was detected by gas chromatography. The ORD spectrum of Fr-I gave a negative Cotton effect which showed a

433 TABLE

II

CHEMICAL

COMPOSITION

OF THE PURIFIED BLOOD

Constituent

gg/mg

#mol/100 mg

Galactose Fucose Glucose Mannose Galactosamine Glucosamine Threonine l>roline Serine Valine Glycine Lysinc Alanine Histidine G l u t a m i c acid lsoleucine Aspar ti~ acid Leucine

128.1 99.0 32.5 13.2 165.6 46.3 74.0 61.1 43.8 23.9 16.9 2.7 2.0 1.6 1.6 1.1 0.8 0.6

71.1 60.3 18.0 7.3 92.4 25.8 62.2 53.1 41.6 20.4 22.5 1.9 2.2 1.1 1.1 0.8 0.6 0.4

GROUP

A-ACTIVE SUBSTANCE

trough at 213 nm ([a] --1 160°), suggesting the presence of N-acetylated hexosamine residues. The molecular weight of Fr-I was estimated by the sedimentation equilibrium, and the value of 100 000 was obtained at both 6000 and 10 000 rev./ min, assuming a partial specific volume of 0.675 which was calculated from the analytical data [19,20]. The purified blood group A-active substance, Fr-I, did not show either B or H activity. Fr-I was found to inhibit completely the agglutination of type A erythrocyte caused by lima bean lectin (4 hemagglutinin dose, 200 pg) at a concentration of 1.3 pg/ml. The interaction of Fr-I to concanavalin A was investigated by the quantitative precipitation method, but Fr-I did not form any precipitate with concanavalin A. Discussion The specific blood group A activity of oyster viscera is particularly interesting, because a few active substances reported for other shellfish had the H activity. Nakazawa [21], and Hayashi and Matsubara [22] reported giycolipids of oyster, but they did not mention the blood group activity of the glycolipids. Glycogen in the neutral fraction was digested by a successive treatment with a-amylase and amyloglucosidase. The s-amylase used in this experiment could digest oyster glycogen to give a single elution peak n e a r K d = 1 in Sepharose 4B chromatography. The lack of contamination of the preparation with glycogen after digestion was shown by the result of polyacrylamide gel electrophoresis in which the standard oyster glycogen remained near the starting position. The presence of glucose was not due to contamination by a glycolipid, since neither fatty acid nor sphingosine base was detected.

434

When the glycogen-free neutral fraction was dissolved in saline solution, some insoluble portion remained. The solubility was improved by a subsequent digestion with Pronase. Donald [23] reported that Pronase treatment of insoluble blood group substances from human ovarian cyst fluids produced a watersoluble active residue which contained all of the original carbohydrates. The most active substance (Fr-I) was recovered from the DEAE-Sephadex column before the gradient elution started. This result agreed with the low mobility of Fr-I in electrophoresis. The polyacrylamide gel electrophoretogram shown in Fig. 3B was obtained by using Tris/borate as an electrode buffer with the period for electrophoresis extended to 2 h to effect the mobility. The glucosamine to galactosamine ratio of 0.6--2.0 for the blood group A substances has been reported for ovarian cyst fluid preparations [24,25]. For the most active blood group A-active fraction from human erythrocyte membrane, this ratio of hexosamines was 0.34 [26]. The ratio (0.28) obtained in the present experiment was low and close to that from erythrocyte membrane. The O R D spectrum of blood group A substances from ovarian cyst fluids showed a pronounced Cotton effect (a trough near 220 nm) which was considered to be associated with a 2-acetamido group [27]. The hexosamine residues in Fr-I were estimated to be N-acetylated. The average molecular weight of blood group substances from ovarian cyst fluids generally ranged from 200 000 to several millions [2]. The value in Fr-I was relatively small. Treatment with Pronase did not markedly decrease the molecular weight of the active substance, because the substance without Pronase digestion behaved similarly to the Pronase-treated substance in sedimentation equilibrium and gel filtration on Sepharose 4B. Concanavalin A is a well-known lectin non-specific to blood group ABO type and cannot agglutinate intact human erythrocytes. Lloyd [28] had demonstrated that concanavalin A could precipitate the blood group A substances from hog stomach linings, while those from ovarian cyst fluids were not precipitated. The present result suggested that the substance from oyster viscera resembled that of ovarian cyst fluids in its terminal nonreducing residues.

Acknowledgements The authors thank Dr. M. Kawakami, Dr. K. Hotta, and Mr. T. Kondo, School of Medicine, Kitasato University, for ultracentrifugal analysis. This work was supported in part by a Grant-in-Aid for Scientific Research (No. 967154) from The Ministry of Education, Science and Culture, of Japan. References 1 L a n d s t e i n e r 0 K. ( 1 9 0 1 ) Wien. Klin. W o c h s c h r . 14, 1 1 3 2 - - 1 1 3 4 2 Watkins0 W.M. ( 1 9 7 2 ) in G l y c o p r o t e i n s ( G o t t s c h a l k , A., e d . ) , 2 n d e d n . , P a r t B, p p . 8 3 0 - - 8 9 1 , Elsevier, Amsterdam 3 I t a s a k a , O. ( 1 9 6 6 ) J. B i o c h e m . T o k y o 6 0 , 4 3 5 - - 4 3 8 4 Gilb, B., Z a h n , I. a n d S c h e i b e , E. ( 1 9 6 7 ) Z. I m m u n i t a t s f o r s c h . AUerg. Klin. I m m u n o l . 1 3 3 , 3 8 5 - - 3 9 3 5 0 g a m o , A., W a t a n a b e , H. and Nagasawa, K . ( 1 9 7 4 ) J. B i o c h e m . T o k y o 76, 5 7 3 - - 5 8 2 6 Whistler, R . T . a n d BeMiHer, J . N . ( 1 9 6 2 ) A r c h . B i o c h e m . B i o p h y s . 9 8 , 1 2 0 - - 1 2 3 7 G a l b r a i t h , W. a n d G o l d s t e i n , I.J. ( 1 9 7 2 ) M e t h o d s E n z y m o l . 28, 3 1 8 - - 3 2 3 8 C l a m p , J . R . . B h a t t i , T. and Chambers, R . E . (19"71) M e t h o d s B i o c h e m . A n a l . 19, 2 2 9 - - 3 4 4

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9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Reinhold, V.N. (1972) Methods Enzymol. 25, 244--249 Warren, L. (1959) J. Biol. Chem. 234, 1971--1975 Ando, S. and Yamakawa, T. (1971) J. Biochem. Tokyo 70, 335--340 Ornstein, L. and Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404---427 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - 4 4 1 2 Arai, K. and Wallace, H.W. (1969) Anal. Biochem. 31, 71--76 Zacharis, R.H., Zell, E.T., Morrison, J.H. and Woodlock, J.T. (1969) Anal. Biochem. 30, 148--152 Yphantis, D.A. (1960) Ann. N.Y. Acad. Sci. 88, 586---601 So, L.L. and Goldstein, I.J. (1967) J. Biol. Chem. 242, 1617--1622 Dubois, M., Gilles, K.A., Hamilton, J.K., Robers, P.A. and Smith, F. (1956) Anal. Chem. 28, 350-356 Gibbons, R.A. (1966) in Glycoproteins (Gottsehalk, A., ed.), 2nd edn., Part A, pp. 29---95, Elsevier, Amsterdam Schachman, H.K. (1957) Methods Enzymol. 4, 32--103 Nakazawa, Y. (1959) J. Bioehem. Tokyo 46, 1579--1585 Hayashi, A. and Matsubara, T. (1969) J. Bioehem. Toky o 65, 503--511 Donald, A.S.R. (1973) Biochim. Biophys. Acta 317,420---436 Rondle, C.J.M. and Morgan, W.T.J. (1955) Bioehem. J. 59, xiii--xiv Hiyama, N. (1962) in Biochemistry and Medicine of Mucopolysaccharides (Egami, F. and Oshima, Y., eds.), pp. 161--182, Maruzen, Tokyo Yamato, K., Handa, S. and Yamakawa, T. (1975) J. Biochem. Tokyo 78, 1207--1214 Beychok, S. and Kabat, E.A. (1965) Biochemistry 4, 2565--2574 Lloyd, K.O., Kabat, E.A. and Beychok, S. (1969) J. Immunol . 102, 1354--1362