Comparative studies of human, equine, porcine and bovine erythrocyte membrane sialoglycoproteins

Comp. Biochem. Physiol., 1976, Vol. 55B, pp. 37 to 44. Pergamon Press. Printed in Great Britain

COMPARATIVE STUDIES OF HUMAN, EQUINE, PORCINE AND BOVINE ERYTHROCYTE MEMBRANE SIALOGLYCOPROTEINS HIDEAKI HAMAZAKI,KYOKO HOTTA.1 AND KAZUHIKOKONISHI2 1Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan and 2Biological Institute, Faculty of Science Tohoku University, Sendai, Japan

(Received 8 October 1975) Abstract--1. Major sialoglycoproteins were isolated from human, equine, porcine, and bovine red cell membranes. 2. SDS-polyacrylamide gel electrophoresis of the sialoglycoproteins following cyanogen bromide treatment showed that carbohydrates were localized on the limited peptide regions of these sialoglycoproteins. 3. The carbohydrate contents of the human, the equine, the porcine, and the bovine sialoglycoproteins were 60%, 40%, 65%, and 80%, respectively. 4. Hemagglutination inhibition tests suggested that the structural similarity of carbohydrate chains was present between the human and the equine sialoglycoproteins.

INTRODUCTION

Giycoproteins comprise approximately 10% of the total protein of the human erythrocyte membrane, with sialoglycoprotein being predominant. The latter, at least 60% of whose molecular mass is carbohydrate, has exhibited A, B and MN blood group activities as well as receptor sites for influenza viruses and various phytohemagglutinins (Marchesi et al., 1971). Although the human erythrocyte membrane sialoglycoprotein has been studied extensively (Marchesi & Andrews, 1971; Fukuda & Osawa, 1973; Javid & Winzler, 1974), those from blood cells of other species have remained obscure (Kobylka et al., 1972). Elucidation of their variations can provide a basis for understanding the structural characteristics of the erythrocyte membrane. This report summarizes a comparative study of the major sialoglycoproteins of human, equine, porcine, and bovine erythrocyte membranes and describes the species uniqueness of these glycoproteins. EXPERIMENTAL PROCEDURES

Erythrocyte membrane and sialoglycoprotein preparation Outdated A, B, and O human bloods were obtained from the Kitasato University Hospital Blood Bank. Horse blood was obtained from the Research Center for Veterinary Sciences of Kitasato Institute. Porcine and bovine bloods were collected at a commercial slaughter house in Sagamihara. The animal bloods were collected into EDTA-Na2 solution (2mg/ml

blood). All blood cell fractions were washed five times in pH 7.2, 5 mM phosphate buffer containing 0.15 M NaC1 (PBSt). The erythrocyte membranes from all four species were prepared by the procedure of Dodge et al. (1963). Crude membrane sialoglycoprotein was isolated by the method of Kathan & Winzler (1961), which calls for the addition of an equal volume of phenol to a 2-5% suspension of erythrocyte membranes in PBS, and then heating of the mixture at 67°C for 10 rain, cooling to room temperature and centrifugation. Phenol extraction of the lower phase was repeated twice. Dialysis of the aqueous layer against water preceded lyophilization. Aliquots of about 20 mg of lyophilized material were then dissolved in 10 ml of 0.01 M Tris-HC1 buffer pH 7.9 and chromatographed on a DEAE-cellulose column (Whatman DE-32, 1.6 x 15 cm) equilibrated with the same buffer. After application of the sample, an additional 50 ml of the same buffer were passed through the column. A 400 ml linear gradient was used to elute a column with 0.01 M Tris-HC1 buffer, pH 7.9 and containing 2 M NaC1, as the limiting buffer. Fractions of about 10ml were collected and analyzed using the thiobarbituric acid reaction for sialic acid and 278 nm. Main fractions containing sialic acid were pooled, dialyzed and dried by lyophilization. Each species whole blood sample of about 200 ml yielded approximately 25 mg of purified sialoglycoprotein.

Polyacrylamide gel electrophoresis Gels containing 10% acrylamide and 0.1% sodium dodecyl sulfate were prepared, run and stained according to the method of Segrest (Segrest & Jackson, 1972). Samples were dissolved in 0.01 M phos*To whom correspondence should be addressed. t Abbreviations used in this report are: PBS, 5mM phate buffer containing 1% SDS, 20% glycerol and phosphate buffer, pH 7.2, containing 0.15 M NaC1; Con 5% 2-mercaptoethanol and heated at 100 ° for 2 min. A, concanavalin A; H-PHAP, purified Phaseolus vulgaris The gels were subsequently stained with Coomassie phytohemagglutinin; PAS, periodic acid-Schiff; SDS, Blue to reveal the protein bands. Carbohydrate consodium dodecyl sulfate. taining material was located using the PAS pro37

38

HIDEAKI HAMAZAKI,KYOKO HOTTA AND KAZUHIKOKONISHI

cedure. All reported gel scans were obtained with the Gilford 2400S spectrophotometer equipped with a model 2410 S linear transport, scanning at 590 nm for Coomassie Blue and at 543 nm for PAS. The following proteins (Schwartz Bio-Research) and their molecular weights were used as standards to determine molecular weights of the glycoproteins under study: bovine serum albumin, 67,000; human immunoglobulin H-chain, 58,000; ovalbumin, 45,000; chymotrypsinogen A, 25,000; and human immunoglobulin L-chain, 22,000. Molecular weights were derived by calibration of the gels with the peptides of known molecular weight and carried out at 22 '~' according to Weber & Osborn (1969).

Analytical methods Total hexose was measured by the phenol-H2SO4 method (Dubois et al., 1956) using a galactose standard. Sialic acid was determined using Warren's thiobarbituric acid procedure (Warren, 1959) and N-acetylneuraminic acid (Sigma Chemical Co.) as standard. Analysis for hexosamines was based on the method of Elson and Morgan as described by Boas after hydrolysis of the material in 4N HCI at 100~C for 6 hr and treatment described previously (Hotta et al., 1970). Analyses on the amino acid analyzer (Hitachi-034) were also performed for the hexosamines. Protein was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. Amino acid analyses Samples were hydrolyzed in glass-distilled, constant boiling HC1 at a concentration of l m g per 0.5 ml in sealed, evacuated tubes at 10TC for 20 hr. Analyses were performed on Nihon-Denshi JLC 6AH (Tokyo, Japan) amino acid analyzer. Gas-liquid chromatograph), Gas-liquid chromatography was carried out with Nihon-Denshi model JGC-1100 (Tokyo, Japan) equipped with dual flame ionization detectors and nitrogen carrier gas. Sugars were chromatographed as their trimethylsilyl derivatives after preparation according to Sweeley et al. (1963). Hernagglutination and hemmagglutination inhibition tests Concanavalin A was obtained from Calbiochem and used directly. Difco Phaseolus vulgaris phytohemagglutinin (PHAP) was further purified with the method of Allen et al. (1969). The purified PHAP (H-PHAP) was homogeneous electrophoretically and used in this study. Serial twofold dilutions of phytohemagglutinins or antisera were made in PBS using the microtitrator apparatus (Cooke Laboratory Product, Alexandria, Va.). Washed human A, B and O and animal erythrocytes suspended (4%) in PBS were added to the diluted solution and the plates were sealed and incubated for 30 rain at toom temperature. The agglutination titer (units of agglutinin activity) was considered to be the reciprocal of the highest dilution of sample that gave positive agglutination. Rabbit anti-M and anti-N serum (Ortho Pharmaceutical, Rariton, New Jersey) were used for the test. Antisera A and B were obtained from Midori-Juji (Osaka, Japan). Hemagglu-

tination inhibition was determined with serial 2-fold dilutions of sialoglycoproteins in PBS. Hemagglutination inhibitory activity is defined as the least quantity of the hexose contents which prevent the agglutination of 25 #l of a 4°41 suspension of erythrocytes by the amount of antiserum of phytohemagglutinins capable of inhibiting the agglutination.

Treatment with cyanogen bromide One mg of sialoglycoprotein was dissolved in 0.1 ml of 50°~, formic acid. To this was added 0.1 ml of 0.31~iicyanogen bromide dissolved in 90°~ formic acid; the reaction mixture was incubated 2YC. After 48 hr, the mixture was dried in vacuo to remove all trace of formic acid. Degree of cleavage was determined by SDS polyacrylamide gel electrophoresis. Alkali treatment Samples were treated with 0.5 N NaOH at 37 C for varying periods of time from 0 to 3 hr. The absorbancy at 241 nm was recorded automatically using a Gilford 2400S spectrophotometer and plotted for every 20-sec interval. To determine the number of O-glycosidic linkages, losses of serine and threonine in sialoglycoproteins caused by the alkaline treatment were taken. Samples were treated with 0.5 N NaOH containing 1 M solium borohydride at 37'C for 3 hr, after which they were neutralized with 1 N HC1, and reduced by the procedure of Tanaka & Pigman (1965). For the purpose of gel chromatography, 1.5 mg of human sialoglycoprotein, or 2.7mg of bovine sialoglycoprotein, was added to 0.2 ml of 1 M sodium borohydride in 0.5 N NaOH and incubated at 37~'C for 3 hr. The solution was then neutralized with 1 N HC1 and chromatographed on Bio Gel P-30 column (1.0 x 48 cm). The products were eluted with distilled water and assayed for sialic acid using the thiobarbituric acid reaction.

RESULTS

Pur!fication of Sialoglycoproteins Sialoglycoproteins were extracted from human, equine, porcine and bovine erythrocyte ghost membranes with phenol. These crude sialoglycoproteins were applied to columns of DEAE cellulose. The elution diagrams of the sialoglycoproteins are shown in Fig. 1. Human sialoglycoprotein was eluted from the DEAE cellulose chromatography at 0.25M NaC1 concentration and those of the other species were eluted at slightly lower salt concentrations: 0.23 M for equine; 0.20 M for porcine; and 0.18 M for bovine. Sialic acid-rich fractions, designated by the bar in Fig. 1, were pooled, dialyzed and lyophilized. The resulting purified sialoglycoproteins were used for further experimentation. Polyacrylamide gel electrophoresis amt nu)lecular weight determination. Polyacrylamide gel electrophoresis of the sialoglycoproteins in SDS was performed in order to analyze the proteins. Figure 2 shows the carbohydrate and the protein staining profiles of the sialoglycoprotein of the species under study. Each of these shows a single major glycoprotein band with Coomassie Blue

Erythrocyte membrane sialoglycoproteins

0.( - ( a )

~

-

39

(c) .0 A

0.~

0.2 E E

o n

0.4

-Ib,

A . "'t I°,

"'t

0.2

I0

20

0

Frocfion

I0

20

30

number

Fig. 1. DEAf-cellulose chromatography of sialoglycoproteins from different species. Crude material from erythrocyte membranes was dissolved in 10 ml of 0.01 M Tris-HCl buffer (starting buffer). (a) 17.5 mg of human, (b) 27.9 mg of equine, (c) 38 mg of porcine and (d) 37 mg of bovine material were applied to DEAf-cellulose columns (1.6 x 15 cm) equilibrated with the same buffer. The columns were washed with 50 ml of the starting buffer, and gradient elutions were then performed. Fraction of 10 ml were collected and analyzed for sialic acid (O--O) and at 278 nm (O-e). Tubes designated with a bar were pooled. See text for details. and PAS staining and do not indicate a second component except in the case of the porcine, where low molecular components stained only by Coomassie Blue are barely evident. In the case of bovine sialoglycoprotein, the protein was located at the sample-running gel interface (see Fig. 4D). A decrease of 5 ~ in the acrylamide concentration did not affect band mobility. The approximate molecular weights of these sialoglycoproteins were determined by the method of Weber & Osborn (1969). From the results of electrophoresis in the presence of 0.1% SDS, molecular weights of the sialoglycoproteins were calculated to (a)

be as follows: human, 56,000; equine, 44,000; and porcine, 40,000-relative to the standard proteins (Fig. 3). Electrophoretic mobility of the bovine sialoglycoprotein indicated a molecular weight of over 100,000.

Amino acid and carbohydrate analyses Results of the quantitative analyses of the amino acid and carbohydrate components of substances from the four species are given in Table 1. Although dissimilarities in the composition of the sialoglycoproteins were evident, there were notable similarities. Cysteine was absent in all four species. The content of amino acid such as aspartic acid, proline, alanine

(b)

(c)

E

<

CB=, 8

_J

~ 6

4

2

8

6

Migration

4 distance,

2

0

6

4

2

0

cm

Fig. 2. Scans of SDS polyacrylamide gels of sialoglycoproteins from erythrocyte membranes of different species. (A) Human, (B) equine and (C) porcine. Electrophoresis was carried out as described under "Experimental procedures" in 0.1 M phosphate buffer, pH 7.2. The gels contained 10~o polyacrylamide and 0.1% SDS. Gels were stained with Coomassie Blue (CB) and PAS and scanned with a Gilford spectrophotometer equipped with a linear transport.

HIDEAKI HAMAZAKI,KYOKO HOTTA AND KAZUHIKOKONISHI

40

Table 2. Hemagglutination activity of erythrocytes from four species with Con A and H-PHAP vine

serum

albumin

noglobulin

M-chain

Minimum c o n c e n t r a t i o n of h e m a g g l u t i n i n r e q u i r e d to completely agglutinate

×

e r y t h r o c y t e s of Equine

Human

Porcine

Bovine

1250

1250

50

50

Pg

Con A H-PHAP

I

I

I

I

I

I

03

02

05

04

05

06

Relative

07

mobility

Fig. 3. Molecular weights of sialoglycoproteins from different species as determined by gel electrophoresis in polyacrylamide gel in the presence of 0.1% SDS, containing 0.1 M phosphate buffer, pH 7.2. (a) Human, (b) equine and (c) porcine. Fifty micrograms of each protein were applied to the top of the gel. The molecular weights of the standards (O) are: bovine serum albumin (67,000), Immunoglobulin H-chain (58,000), Ovalbumin (45,000), Chymotrypsinogen (25,000) and Immunoglobulin L-chain (22,000). Plot of the logarithm of molecular weight vs mobility relative to that of the tracking dye. The line was applied to the points using the method of least squares. and leucine varied from one species to another. In human sialoglycoprotein, glutaminic acid predominated, followed by aspartic acid, threonine and serinc. Threonine and leucine were the most abundant Table 1. Comparison of the amino acid and carbohydrate compositions of sialoglycoproteins obtained from human. equine, porcine, and bovine erythrocyte membranes Constituents Human

Sialoglycoproteins Equine Porcine Bovine moles / lO000g peptides

Lysine

3.85

2.84

6.20

Histidine

2.75

1.35

1.77

1.04

Arginine

2.23

3.63

1.33

7.21

A s p a r t i c acid

9.76

8.26

8.42

4.66

Threonine

9.48

9.08

8.56

15.50 9.17

Serine

3.88

9.89

8.26

5.91

13.33

7.59

7.83

8.44

Proline

4.67

9.21

7.97

lO.IO

Glycine

7.01

8.67

9.60

5.88

Alanine

5.50

8.53

7.53

6.14

Cysteine

0

0

0

0

Valine

5.50

6.64

6.20

7.07

Methionine

4.26

1.49

6.20

NDa

Isoleucine

4.26

5.96

5.17

3.55

Leucine

4.56

9.21

6.62

5.03

Tyrosine

2.75

1.22

1.33

2.77

Phenylalanine

0.96

2.44

2.22

I.]5

20.31

]2.71

45.78

]02.85

Mannose

8.85

3,31

3.69

1.0

Fucose

2.39

0.60

none

none

N-acetylgalactosamine

7.53

5.58

4.34

II.06

N-acetylgIucosamine

3.85

1.25

9.07

42.10

Glutamic acid

1250

2.5

50

3

amino acids in equine sialoglycoprotein. Porcine material exhibited high glutamic acid, threonine, proline, aspartic acid, alanine and glycine content. Bovine sialoglycoprotein showed a relatively high amount of threonine, proline, leucine, glutamic acid and serine. Human and equine sialoglycoproteins were similar in carbohydrate composition, although the carbohydrate content of the former was much higher (60°.0) than that of equine (40%). In general, galactose and sialic acid were abundant, followed by galactosamine and mannose. Glucosamine and fucose were the least abundant. The carbohydrate composition of porcine and bovine sialoglycoproteins differed greatly from that of the human. Fucose was absent from porcine and bovine sialoglycoproteins, while mannose was almost negligible in the latter; the two contained substantial amount of galactose and glucosamine. The bovine carbohydrate content of 80% was the highest found in the four species; porcine followed with 65°;.

Hemagglutination and hemagglutination inhibiting actirity Hemagglutination titer of Con A and H-PHAP were determined with human blood type A, B, and O, equine, porcine, and bovine erythrocytes (Table 2). The minimum concentrations of Con A and H-PHAP needed to agglutinate equine erythrocytes were 2.5 and 3 Hg per ml, respectively. A weak reactivity of Con A and H-PHAP with porcine erythrocytes was shown. Con A and H-PHAP did not agglutinate bovine erythrocytes at concentrations of less than 1250 and 50pg per ml, respectively. Con A also did not agglutinate human erythrocytes. Table 3 lists the inhibitory activity of the sialoglycoproteins against agglutination of equine erythrocytes by Con A and H-PHAP. There were significant variations with porcine and bovine sialoglycoproteins. Table 3. Hemagglutination inhibiting activity of sialogl3~coproteins obtained from the erythrocyte membranes of four different species Minimum amount of hexose content in sialoglycopPoteins completely i n h i b i t i n g

Galactose

S i a ] i c acid a ND : Not determined. was not s a t i s f i e d .

24,32

6.80

9.]0

27.16

Separation of methionine and hexosamine

f o u r h e m a g g l u t i n a t i n g doses Human

Equine

Porcine

~ovine

7.7

46

Pg Con A

2.7

1.3

H-PHAP

0,33

,9.08

31

0.72

Erythrocyte membrane sialoglycoproteins

(a)

41

(c)

(b)

L

g

8

6

2

4

0

8

6

Migration

4

2

distance,

0

I

8

I

6

I

4

]

2

cm

(d)

"oJ2 2 06 <

0.4

02

o

PAS

CB

I

I

]

I

8

6

4

2

Migration

distance,

cm

Fig. 4. Scans of SDS polyacrylamide gels of cyanogen bromide treated sialoglycoproteins. (a) Human, (b) equine, (c) porcine and (d) bovine. The material was treated with 0.3~o cyanogen bromide in 70~o formic acid at 25° for 48 hr. Electrophoresis containing 10~o polyacrylamide--0.1~o SDS was carried out as described under "Experimental procedures". Gels were stained with Coomassie Blue (CB) and PAS, and absorption was measured with a gel scanner. Bovine sialoglycoprotein (SGP) is shown in Fig. 4d.

The highest activity was exhibited by equine sialoglycoprotein, as was expected, Porcine sialoglycoprotein possessed a trace of Con A activity but not with H-PHAP. Bovine sialoglycoprotein had significant H-PHAP activity. Human sialoglycoproteins from blood type A, MN: B, NN and O, MM erythrocytes were tested against the human blood group. Each sialoglycoprotein had the potent activities of A, B, M and N, depending on the blood groups of the original erythrocytes. Equine sialoglycoprotein had a trace of human blood group A activity when a 250 #g sample was used for the test; however, bovine sialoglycoprotein showed none. Porcine sialoglycoprotein was not tested.

Cyanogen bromide cleavage Sialoglycoproteins from the four species were treated with cyanogen bromide and then analyzed by SDS polyacrylamide gel electrophoresis as shown in Fig. 4. Several unique fragments were obtained from human sialoglycoprotein of blood group O erythrocyte origin. The main fragment stained with Coomassie Blue and PAS. The other fragments, which were PAS negative, appeared to be a simple polypeptides with a tool. wt of about 15,000. The same results were obtained when sialoglycoprotein from human blood group B erythrocytes was examined. The products from equine sialoglycoprotein, two major fragments, are shown in Fig. 4B. Note that the faster fragment

42

HIDEAKI

o ,5~,t~

HAMAZAKI,

KYOKO

(a)

(b)

HOTTA

AND

KAZUHIKO

KONISHI

Table 4. Loss and recovery of amino acid after alkaline, reductive degradation ] t t1111~111

•

0

24

48 Time,

72

a•

96 •

mln

b •

O•

••° •

2 m LI <{

•

C•

d

" i::-"

]:AOD =0.02

I I

Reaction t i m e ,

hr

Fig. 5a, Change in absorbancy with time at 241 nm when sialoglycoproteins were treated with alkali. Absorbancies at 241 nm were recorded automatically with a Gilford spectrometer, a 63/.tg of human, b 133/~g of equine, c 34/~g of bovine and d 55/*g of porcine sialoglycoproteins were treated with 0.5 N NaOH at 37°C for 3 hr. See text for details. Fig. 5(b). Guggenheim plotting of fi-elimination reaction. Difference of absorption at two times of 90 min intervals was plotted against time. a Human. b equine. c bovine and d porcine. designated as (II) stained more intensely with Coomassie Blue than the slower fragment (I). Mobilities of cyanogen bromide treated porcine and bovine sialoglycoproteins were almost equal to that of the intact glycoproteins. Simple peptides of low molecular weight were evident in both cases.

Serind' Alanmc ~ Threonine' z - A m i n o b u t y r i c acid k'

3 iI

~ 2u(~ 3 15 +-206

I qtlinc

I)orcinc

I11/)]¢S 10,0(10 g pcptides 1.37 1f12 - I 5u ~ 1.06 23u 187 ~ 1 71 ~ 142

BO~, inc

3 53 ~ 2 56 v lS e ~67

~'Loss of serine or threonine expressed as moles/10.000 g peptides. h Increase of alanine or >aminobutyric acid expressed as moles/10.000 g peptides. human, 3.2-3.9 mole for equine, 2.5 2.9 mole for porcine, and 6.2-10.7 mole for bovine. In order to determine the size of oligosaccharide chains released from the peptide backbone by alkaline treatment, gel filtrations of the alkali-borohydride treated human and bovine sialoglycoproteins were performed as shown in Fig. 6. The first peak of human sialoglycoprotein which appeared at the void volume contained the residual glycoprotein, and the second peak contained the released oligosaccharides. The third peak consisted of oligosaccharides of smaller molecular than those of the second peak, Results indicated that some of the carbohydrate chains in human sialoglycoprotein are linked to the peptide backbone through an alkali-stable linkage reported previously, such as the N-acylglycosylamine type, and the elimination reaction simply did not go to completion. However, nearly complete elimination was accomplished in the case of bovine sialoglycoprotein (Fig. 6), that is to say, only a trace of material was excluded at the void volume from the gel. This was to be expected after the results described earlier (Fig. 5). Most of the material was shifted to the "Dextran 10" or "'stachiose" region. The three main peaks were evident after alkali-treatment (Fig. 6). Intact sialoglycoproteins were eluted as a sharp at the void volume on the Bio Gel P-30 in all cases. (a) 02

Determination of O-glycosidic linkage The release of unsaturated amino acids as a result of fl-elimination from the sialoglycoproteins was recorded automatically, and the absorbancies at 241 nm were plotted (Fig. 5A). After 3 hr, the reactions on all four species substrates were still incomplete. The Guggenheim plotting (Guggenheim, 1926) determined straight lines, this indicated that fl-elimination was pseudo-first-order reaction (Fig. 5B). The half life period i.e. the time required to halve the cleavage of O-glycosidic linkage under these experimental conditions was obtained from the slope of the linear line (Fig. 5B); in all cases it was 52 rain. It was assumed that 91% of O-glycosidic linkage was destroyed in 3 hr. Number of O-glycosidic linkages in a molecule was calculated on the basis of the losses of serine and threonine and of the recoveries of alanine and a-aminobutyric acid after alkaline, reducrive degradation (Table 4). The following molar values (per 10,000 g peptide) of sialoglycoprotein O-glycosidically linked units were obtained: 4.1~5.3 mole for

u~

g 0 (b)

2 q

Void

Dextran

IO

Sfaeh,ose

02

'3 I

I0

20 Frachon

30

number

Fig. 6. Gel filtration of alkaline borohydride treated human and bovine sialoglycoproteins. (A) 1.5 mg of human sialoglycoprotein and (B) 2.7 mg of bovine sialoglycoprotein were treated with alkali-borohydride, as described in the text, for three hours and then applied to a Bio Gel P-30 column (1.0 x 48 cm). Elution was performed with distilled water. Fractions of 1.1 ml were collected. Fractions were analyzed for sialic acid by the Warren method.

Erythrocyte membrane sialoglycoproteins DISCUSSION Isolation of major sialoglycoproteins from human, equine, porcine and bovine erythrocyte membranes was achieved by a phenol extraction method followed by DEAE-cellulose column chromatography. Sialoglycoproteins thus obtained appeared almost homogeneous on SDS polyacrylamide gel electrophoresis. A molecular weight of 55,000 has been reported for the major glycoprotein from human erythrocyte membranes (Segrest et al., 1971), and from its molecular weight and chemical composition, our preparation of human erythrocyte origin could be considered the same as their glycoprotein. The molecular weight of the sialoglycoproteins from different species varied from 40,000 to over 100,000. Although it is reported that the molecular weight of bovine major glycoprotein varied from 180,000 to over 200,000 (Kobylka et al., 1972), the problem of determining the molecular weights of sialoglycoproteins by SDS polyacrylamide electrophoresis remains, since bovine sialoglycoprotein carries a large amount of carbohydrate (about 8070). Each species investigated appears to have a single major sialoglycoprotein, each with somewhat different characteristics. The peptide size in the sialoglycoprotein was calculated to be 22,000 for human, 26,000 for equine and 15,000 for porcine according to the molecular weight and the protein content of each sialoglycoprotein. None of the sialoglycoproteins were found to contain cysteine. This indicates that the sialoglycoproteins from the four species are composed of a single polypeptide chain as reported earlier for the human glycoprotein. On the basis of the results obtained, the following can be stated about the sialoglycoprotein carbohydrate chain structure: (1) The carbohydrate chain of equine sialoglycoprotein seems to be similar to that of the human, as deduced from its chemical composition, from the strong inhibitory activity against Con A and H-PHAP, and from the existence of both O-glycosidically and N-glycosidically linked oligosaccharide chains. Mannose residues are not linked O-glycosidically to the serine or threonine residues of the peptide backbone (Thomas & Winzler, 1969). (2) Porcine sialoglycoprotein carries the receptor for only Con A, though the activity is very low as compared to its human counterpart. The existence of a glycosamine-type linkage in porcine sialoglycoprotein is also suggested by the mannose residue. It is proposed that the structural feature of the oligosaccharide in porcine sialoglycoprotein are considerably different from the human sialoglycoprotein. (3) Bovine sialoglycoprotein does not have the receptor for Con A. This result is consistent with the finding of no mannose in bovine sialoglycoprotein, since the specificity for binding to the Con A was shown to reside with internal units of ~-D-mannose containing oligosaccharide (So & Goldstein, 1968). On the other hand, bovine sialoglycoprotein bound to H-PHAP in spite of the lack of mannose. The structural features of the oligosaccharide which determined PHAP binding were shown to be the galactose residues in the outer branches of the molecular and the mannose residues in the core (Kornfeld & Kornfeld, 1970). This fact suggests the possibility that the mannose residues

43

can be substitutes for similar monosaccharide residues. From the lack of any evidence of mannose and from the pattern of gel chromatography after alkali treatment of bovine sialoglycoprotein, it can be concluded that oligosaccharide chains of the bovine sialoglycoprotein are composed of only O-glycosidically linked oligosaccharide chains. In glycoproteins whose serine and/or threonine are involved in the carbohydrate-peptide bond, the amino acid residue may be recognized by fl-elimination which occurs when the glycoprotein is subjected to mild alkali treatment. Percentages of the serine and threonine residues involved in O-glycosidic linkages with carbohydrate were as follows; human 21-33~o, porcine 17-20%, equine 19-23% and bovine 28~4~o. Considering the orientation of the glycoprotein at the cell surface and the arrangement of its oligosaccharide moieties, it has been proposed that the major sialoglycoprotein of the human red cell is organized in the membrane with its oligosaccharide-rich portions exposed to the exterior environment of the cell (Jackson et al., 1973). From the cyanogen bromide cleavage results, it is concluded that the human glycoprotein is a single polypeptide with a unique amphipathic molecular topography (Segrect et al., 1973). Our data from cyanogen bromide cleavage of the four sialoglycoproteins demonstrated that there were carbohydrate-containing regions and a peptide portion containing no carbohydrate, although the exact order and number of cyanogen bromide breakdown fragments for each sialoglycoprotein cannot as yet be stated. The size of oligosaccharide chain, the order and the structure of carbohydrates and the number of the chains should be different in the four species. SUMMARY

Erythrocyte membrane sialoglycoproteins of human, equine, porcine and bovine origin were isolated by means of phenol extraction and subsequent DEAE-cellulose chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, chemical analyses, cyanogen bromide cleavage, alkali treatment and biological tests were used to establish species sialoglycoprotein differences. Polyacrylamide disc eletrophoretic procedures afforded the following molecular weights for the sialoglycoproteins from the four species: 56,000 for human, 44,000 for equine, 40,000 for porcine and over 100,000 for bovine. Chemical analyses indicated a human sialoglycoprotein carbohydrate content of 60%, as compared to 40~o, 65%, and 8070 found in equine, porcine and bovine derivatives, respectively. Human sialoglycoprotein exhibited multiple blood group antigenicity as well as the specific receptors for concanavalin A and Phaseolus vulgaris phytohemagglutinin. Equine sialoglycoprotein displayed a weak blood group A antigen and the receptors for both plant agglutinins. The porcine counterpart exhibited the presence of Con A receptor, whereas the bovine derivative carried only H-PHAP receptor. Cyanogen bromide cleavage of both native human and equine sialoglycoproteins elicited several fragments, which were demonstrable with polyacrylamide disc gel electrophoresis. Of the human derivatives, one fragment contained most of the molecular carbo-

44

HIDEAKI HAMAZAKI, KYOKO HOTTA AND KAZUHIKO KONISHI

hydrate, whereas two fragments of equine derivative contained the carbohydrate. Mobility of the major fragments resulting from treatment of porcine and bovine sialoglycoproteins showed little differentiation, and several peptide fragments were evident. Results suggest that the erythrocyte m e m b r a n e sialoglycoproteins of each species are distinctive.

Acknowledgements--The authors are indebted to Dr. M. Kurokawa for his encouragement and support. We express our appreciation to Dr. M. Sagawa for his generous gift of equine blood, to Miss Y. Tsujino for the gas-liquid chromatography and to the staffs of Nihon-Denshi for the amino acid analyses.

REFERENCES

ALLEN L. W., SVENSONR. H. & YACHNIN S. (1969) Purification of mitogenic proteins derived from Phaseolus vulgaris" isolation of potent and weak phytohemagglutinins possessing mitogenic activity. Proc. natn. Acad. Sci., U.S.A. 63, 334-341. DODGE J. T., MITCHELL C. & HANAHAN D. J. (1963) The preparation and chemical characteristics of hemoglobinfree ghosts of human erythrocytes. Archs Biochem. Biophys. 100, 119-130. DUBOIS M., GILLES K. A., HAMILTON J. K., REBERS P. A. & SMITH F. (1956) Colorimetric method for determination of sugar and related substances. Anal. Chem. 28, 35(~356. FUKUDA M. & OSAWA T. (1973) Isolation and characterization of a glycoprotein from human erythrocyte membrane. J. Biol. Chem. 248, 5100-5105. GUGGENHEIME. A. (1926) On the determination of the velocity constant of a unimolecular reaction. Phil. Ma9. 2, 538 543. HOTTA K., HAMAZAK1 H., KUROKAWA M. & [SAKA S. (1970) Isolation and properties of a new type of sialopolysaccharide-protein complex from the jelly coat of sea urchin eggs. J. Biol. Chem. 245, 5434-5440. JACKSON R. L., SEGREST J. P., KAHANE I. & MARCHESI V. T. (1973) Studies on the major sialoglycoprotein of the human red cell membrane. Isolation and characterization of tryptic glycopeptides. Biochemistry 12, 3131 3138. JAVID J. I. & WINZLER R. (1974) Association of glycoproreins with the membranes. 1. Isolation and molecular weight of the monomeric unit of the major glycoprotein from human erythrocytes. Biochemistry 13, 3635-3638.

KATHAN R. H., WINZLER R. J. & JOHNSON C. A. (1961) Preparation of an inhibitor of viral hemagglutination from human erythrocytes. J. exp. Med. 113, 37-45. KOBYLKA D., KHETTRY A., SHIN B. C. & CARRAWAY K. L. (1972) Proteins and glycoproteins of the erythrocyte membrane. Archs Biochem. Biophys. 148, 475-487. KORNFELD R. • KORNFELD S. (1970) The structure of a phytohemagglutinin receptor site from human erythrocytes. J. Biol. Chem. 245, 2536-2545. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265 275. MARCHES1 V. T. & ANDREWS E. P. (1971) Glycoproteins: isolation from cell membranes with lithium diiodosalicylate. Science, N . Y 174, 1247 1248. MARCHESI V. T., TILLACK T. W. & SCOTT R. E. (1971) The rote of glycoproteins in red celt membrane structure. In Gl)coproteins of Blood ('ells and Plasma (Edited by JAMIESON G. A. & GREENWALT T. J.) pp. 94 105. J. B. Lippincotk Philadelphia. SEGREST J. P., JACKSON R. L., ANDREWS E. P. 8~ MARCHESI V. T. (1971) Human erythrocyte membrane glycoprotein: a re-evaluation of the molecular weight as determined by SDS polyacrylamide gel electrophoresis. Bioehem. hiophys. Res. Commun. 44, 390 395. SEGREST J. P. & JACKSON R. L. (1972) Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In Methods in Enzymalogy (Edited by G1NSBERG V.), Vol. 28, pp. 54 63. Academic Press, New York. SEGREST J. P., KAHANE I., JACK~)N R. L. & MARCHESI V. T. (1973) Major glycoprotein of the human erythrocyte membrane: evidence for an amphipathic molecular structure. Archs Bioehem. Biophys. 155, 167 183. So L. L. & GOLDSTEIN l. J. [1968) Protein--carbohydrate interaction XIII. The interaction of Concanavalin A with :~-Mannans from a variety of microorganisms. J. Biol. Chem. 243, 2003- 2007. SWEELEY C. C., BENTLEY R., MAKITA M. & WELLS W. W. (1963) Ga~qiquid chromatography of trimethylsilyl derivatives of sugars and related substances. J. Am. Chem. Soc. 85, 2497 2507. TANAKA K. & PIGMAN W. (1965) Improvements in hydrogenation procedure for demonstration of O-threonine glycosidic linkages in bovine submaxillary mucin. J. Biol. ChenL 240, 1487 1488. THOMAS D. B. & WINZLER R. J. (1969) Structural studies on human erythrocyte glycoproteins. Alkali-labile oligosaccharides. J. Biol. Chem. 244, 5943-5946. WARREN L. (1959) The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234, 1971 1975. WEBER K. ~ OSBORN M. (19691 The reliability of molecular weight determinations by dodecyt sulfate--polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412.