Isolation and characterization of a blood group active glycopeptide from porcine aorta

ISOLATION AND CHARACTERIZATION BLOOD GROUP ACTIVE GLYCOPEPTIDE PORCINE AORTA JUNICHIRO

AIKAWA’.‘,

MAMORU

ISEMURA I*, HIROSHI

KEIYA TADA’ and ‘Department

of Biochemistry

and ‘Department 2-l Seiryo-machi.

MANAKA-FA’.

OF A FROM

MASAKI

of Pediatrics, Tohoku University Sendai, Miyagi 980. Japan

(Received

KIKWHI’,

ZENSAK~J YWZAWA’

8 Murch

School

of Medicine.

1985)

Abstract-l. A fucose-rich glycopeptide was prepared from the pronase digest of porcine thoracic aorta by gel-filtration through Sephadex G-100, DEAE-Sephadex A-25 column chromatography and z-amylase digestion. 2. This glycopeptide was electrophoretically homogeneous. 3. The large molecular size and chemical composition suggested that this glycopeptide was derived from mucin-type glycoprotein. The results of the p-elimination reaction indicated that this glycopeptide contained the O-glycosidic linkages between galactosamine and serineithreonine. 4. This glycopeptide exhibited blood group A and H activities, 5. The present study revealed that the porcine thoracic aorta contains a blood group antigen of mucin-type glycoprotein nature

previously (Aikawa et al., 1984a). Four volumes of ethanol containing 1% potassium acetate were added to the supematant with stirring. The mixture was left to stand overnight and then centrifuged at 8000 rpm for 30 min. The sediment was dissolved in water (50 ml). The solution was dialyzed exhaustively against water and lyophilized, yielding a crude glycoconjugate fraction. This fraction was dissolved in water and aqueous loo/;, CPC was added to this solution with stirring to give a final concentration of 1%. The mixture was allowed to stand at room temperature for 1 hr. To the supernatant obtained by centrifugation were added four volumes of ethanol containing 1% potassium acetate and the mixture was left to stand for 16 hr. The mixture was centrifuged at 8000 rpm for 30 min and the sediment was dissolved in water. The solution was dialyzed against water and lyophilized, yielding CPCnonprecipitable fraction.

INTRODUCTION

(7%) as described

Arterial

tissues have been shown to contain various glycoproteins (Barnes and Partridge, 1968; Saito and Yosizawa, 1975; Saito et al., 1975; Roberts and Wagh, 1976; Heifetz et al., 1982; Bressan et al., 1983; Moczar et al., 1983). A recent paper reported the synthesis of lactosaminoglycan structures by vascular endothehal cells in culture (Heifetz et al., 1984). However, to our knowledge, there has been no precise information whether or not the arterial tissues contain a blood group antigen of glycoprotein nature, although lactosaminoglycans may possibly have such activities (Fukuda et al., 1981). In the present paper, we describe that the porcine thoracic aorta contains a fucose-rich mucin (fucomucin)-type glycoprotein with blood group activities.

MATERIALS

Gel-filtration

AND METHODS

r-Amylase immobilized on polyacrylamide beads was purchased from Sigma Chemicals. Porcine aortas and other materials were from the same sources described previously (Aikawa er al., 1984a,b). Preparation

of CPC-soluble

.fraclion

yrom

porcine

aortas

Porcine thoracic aortas were freed from adventitia and surrounding connective tissues. Intima-media (223 g, wet weight) were digested exhaustively with pronase (4.6g in total), after extraction of saline-soluble materials and fat, as described previously (Aikawa et al., 1984a). Following procedures were performed at 4°C unless otherwise stated. The pronase digest was treated with cold trichloroacetic acid

*Author

to whom correspondence CPC+etylpyridinium sodium dodecyl sulfate.

Abbreviations:

should be addressed. chloride; SDS-

through

Sephadex

G- 100

The CPC-nonprecipitable fraction (188 mg) was dissolved in 0.1 M ammonium bicarbonate (pH 8.0) and the solution was applied onto a column (2.5 x 50 cm) of Sephadex G-100 pre-equilibrated with the same solution. Elution was performed with the same solution and fractions of 5 ml were collected. The hexose content and the absorbance at 230 nm of each fraction were determined. The void volume fraction (fraction 2432) and the retarded fraction (fraction 33-80) were separately collected and lyophilized. The former was designated as fraction A. DEA E-Sephadex

A -25 column

chromatography

Fraction A (45 mg) was loaded onto a column (1.5 x 26 cm) of DEAE-Sephadex A-25 (Cl- form) and stepwise elution was carried out with water, then 0.1, 0.2, and 2.0 M NaCl solutions as described previously (Aikawa et al., 1984b). Fractions of 10 ml were collected and monitored for the hexose content and the absorbance at 230 nm. The fractions eluted with water, then 0.1, 0.2, and 2.0 M NaCl solutions were separately collected, dialyzed against water and lyophilized, yielding fractions Al, A2, A3. and A4. respectively. II55

1

-A n2Jh.w~ d@\ riot1

The major glycopeptidc fraction (A?). which was eluted with 0.1 M NaCl from the DEAE-Sephadex A-25 column. was digested with z-amylase immobilized on polyacrylamide beads to remove contammated glycogen as described by Endo and Yosirawa (1973). The digest was separated on a column of Sephadex G-100 under the conditions described above. The fraction eluted at the void volume was designated as fraction 9.

3-

j2 i

The molecular size of fraction B was estimated by gelfiltration through Sepharose CL-49 (I .5 x 153 cm) preequilibrated with 0.5 M ammomum bicarbonate (pH 8.0). Elution was performed with the same solution and K,,, was determined by referring to the elution positions of blue dextrdn (M, = 2.000.000) and glucose (M, = 180). Ferritin (M, = 750,000) and bovine serum albumin (M, = 65.000) were also examined for comparison.

Electrophoresis on cellulose acetate membrane (Separax) was performed as described previously (Munakata ef al., 1980). SDS-?‘,, Polyacrylamide gel electrophoresis and SDS-I”,, agarose gel elcctrophoresis were carried out according to the methods described by Weber and Osborn (1969) and Holden zf 01. (1971). respectively.

Hexose (Dubois (‘I ~1.. 1956). aiahc acid (Warren. 1959). and sulfate (Abdul and Robert. 1978) were determined by the methods reported in the cited references as described previously (Aikawa et al.. 1984~). Individual neutral sugars were determined by gas-liquid chromatography as described previously (Aikawa PI al.. I984b). Hexosamines and amino acids were quantified with a Hitachi amino acid analyzer (Aikawa e( ~1.. 1984b). [j -Elimincrrion

wac,ion

Fraction B (I .8 mg) was dissolved m 0.5 ml of 0.1 M NaOH (Spiro. 1972) containing I M NaBH, and 0.01 M PdCI,. which was prepared according to the procedure described by Tanaka and Pigman (1965). The mixture was kept at 25 C for 24 hr and then filtered to remove PdCl:. A portion of the clear solution thus obtained was applied onto a column (1.0 x I53 cm) of Sephadex G-100 preequilibrated with 0.1 M ammonium bicarbonate. Fractions of 2 ml were collected and monitored for the hexose content. The remaining portion was further divided into two portions. One was hydrolyzed with 4 M HCI at 100 C for 6 hr to determine the contents of hexosamine and hexosaminitol, and the other was hydrolyzed with 6 M HCI at I IO’C for 24 hr for amino acid analysis.

Blood group A, 9, H. and Lewis activities mined by the conventional (Isemura ef a/.. 1983).

methods

as described

were deterpreviously

RESULTS

Isolation

of‘

a

.$-action (fracfion

high B)

molecular

weigh1

glycopeptide

Pronase digestion of 223 g (wet weight) of fresh porcine aortas yielded 1.0 g of the crude glycoconjugate fraction. This fraction was then separated into CPC-precipitable (554mg) and CPCnonprecipitable (188 mg) fractions. Gel-filtration through a Sephadex G-100 column of the CPCnonprecipitable fraction (180 mg) gave the void volume fraction with high molecular weight (fraction A,

2’

ow0

.

I 20

I 40

Froct~on

I 60

1 80

1 100

number

Fig. I. Gel-filtration through a Sephadcx G-100 column 01 CPC-nonprecipitable fraction from the intima-media 01 porcine thoracic aortas. Sample was applied onto a column (2.5 x 50cm) of Sephadex G-100 prc-equilibrated with 0.1 M ammonium bicarbonate (pH 8.0). Elutlon was performed with the same solution. Fractions of 5 ml ucrc collected and monitored for the absorbance at 230 nm ( l ) and the hexose content at 490nm in the phenol-H,SO, reaction (0). The void volume fraction (fractions 24 32) was designated as fraction A. The elution volumes of Blue Dextran ( V,,) and glucose (I’, I are indidcated.

45 mg) and the retarded fraction (91 mg). representing a total recovery of 76’:,, (Fig. I). Fraction A was further separated by DEAE-Sephadex A-25 column chromatography into four fractions (Fig. 2). The yields of fractions Al, A2, A3, and A4 were 25. I 1 11.2, 2.7, and 2.6 mg. respectively, from 45 mg 01 fraction A. Gel-filtration through Sephadex G-100 of the a-amylase digest of fractions A2 (I 1.2 mg) yielded 6.9 mg of fraction B (Fig. 3). Characterization

qf’jlaction

B

On cellulose acetate membrane electrophoresis. fraction B gave a single band either at pH 3.0 or at pH 8.6, when detected by the periodic acid-SchitT reaction. The band was, however, unstainable with Alcian Blue. Fraction B also gave a single band on SDS-I”,, agarose gel electrophoresis as a periodic acid-schiffpositive one (Fig. 5). No effect of reduction with 2-mercaptoethanol suggested that Fraction B was devoid of intermolecular disulfide bridge (Fig. 5). Fraction B did not penetrate into 3:;) polyacrylamide gel, suggesting a very large molecular size of the glycopeptide (Fig. 5). The fact that fraction B retained high molecular weight in spite of the exhaustive digestion with pronase was further demonstrated by gel-filtration through Sepharose CL-4B. Fraction B gave a single peak positive to the phenol-H,SO, reaction with a K,, value of 0.24 (data not shown). Thus the estimated molecular weight of the glycopeptide was about 1,200,000, which was much higher than that of ferritin (750,000) with a K,, value of 0.46. The compositional analyses of fraction B showed that galactose. galactosamine, glucosamine. and

Blood group

20

0

40 Fraction number

60

I IS7

active glycopeptide

The changes in the amino acid composition before and after the p-elimination reaction are listed in Table 3. The contents of serine and threonine were greatly diminished and the alanine content increased after the reaction. A new peak was also observed on a chart of amino acid analysis (data not shown) and identified as r-aminobutyric acid (Table 3). These findings indicated that the hydroxyl groups of seryl and threonyl residue were inv,olved in the glycopeptide linkages. In order to determine the monosaccharide(s) involved in the glycopeptide linkages, a portion of the reaction mixture was analyzed for the contents of amino sugars and their reaction products (Table 3). While the glucosamine content remained unchanged, the galactosamine content was decrcased markedly. The increase in the galactosaminitol compensated this decrease (Table 3). These data indicated that N-acetylgalactosamine was involved in the glycopeptide linkages.

80

Fig 2. DEAE-Sephadex A-25 column chromatography of fraction A. Sample was loaded onto a column (I .5 x 26 cm) of DEAE-Sephadex A-25 (Cl form) and stepwise elution was carried out with water. then 0. I. 0.2. and 2.0 M NaCl solutions. Fractions of IO ml were collected and monitored for the absorbance at 230nm (0) and the hexose content at 490 nm in the phenol-HSO, reaction (0). At arrows I. 2, 3. and 4, elutions were started with H,O. 0.1. 0.2. and 2.0 M NaCI. respectively. and fractions indicated by bars were collected.

Fraction B exhibited strong blood group A activity and weak H activity. The minimum concentrations required to inhibit hemagglutination of blood group A and H erythrocytes were 7.8 and 62.5 pg/ml,

v, 3-

fucosc were the major carbohydrate constituents (Table I) and that thrconine. proline. glutamic acid. and serine were the major amino acids (Table 2), suggesting that fraction B was a mucin-type glycopeptide. Neither sialic acid nor sulfate was detected. These findings were compatible with the electrophoretic observations (Fig. 4), indicating that this glycopeptide was derived from a fucomucin-type glycoprotein. [j-Eliminution

z

; <

0l- . . ..a “..@ ,‘:

I. Chemical

,JY

* . . . . +J‘L..**(

... .. ,

-B

Since the chemical composition of fraction B suggested the presence of the 0-glycosidic linkages between carbohydrate and hydroxyamino acids, which are characteristic in mucin-type glycoprotein, this glycopeptide was subjected to the p-elimination reaction with alkaline borohydride in the presence of PdCI, (Tanaka and Pigman, 1965). The reaction products gave retarded, polydisperse peaks containing carbohydrates on gel-filtration through Sephadex G-100 (Fig. 6). The result suggested that the carbohydrate chains were attached to the polypeptide portion via the alkaline-labile 0-glycosidic linkages.

0

20

40 Fraction

60

80

number

Fig. 3. Sephadex G-100 column chromatography of fraction A2 after z-amylase digestion. Sample was applied onto a column (2.5 x 50 cm) of Sephadex G-100 pre-equilibrated with 0.1 M ammonium bicarbonate (pH X.0). Elution was performed with the same solution. Fractions of 5 ml were collected and monitored for the absorbance at 230 nm (0) and the hexose content at 490nm in the phenolLH,SO, reaction (0). The fraction eluted at the void volume was designated as fraction B. V,, and V,, see Fig. I.

composition

FUCC&

Mannose”

Ci&XtOStZ”

Glucose”

GlUCOS~llllll~~

144

6.0

226

60

I59

Data, expressed “Determined by hDetermined by ‘Determmed as “Determined by ‘Determined by ‘Not detectable

2-

2

reaction

Table

1:

1

1

of fraction

as ~g’mg dry welght. are from duplicate determmations. gas liquid chromatography (Aikawa V, ol.. 1984b). amino acid analyzer (Aikawa r,
B

Galactosamuv? 206

1959).

Slalic acid’

Sulfate”

Ammo acid’

ND’

ND’

I05

JUNKHIKO

115x Table Ammo

2. Amino

acid

composition

Asp&c

of frxtion

acid

73

Threonine

il.8

Serine

148

Glutamic

acid

14.Y

Proline

lb.7

Glycine

6X 10

Alanine Isoleucine LeUClne

34 2 0

Phenylalamne

0 Y

Lysine

I5

Arglnlne

14

No

correctmn

was

made

for

degradation

during

tyrosme.

hlstidine

hydrolysis. Half-cystine. and

valine,

tryptophan

methlonine. were

not

t’/

ul.

1982; Bressan et d.. 1983; Moczar r~r~1.. 1983). Little information is. however. available for mucin-type glycoprotcin of this tissue. although Saito and Yosizawa (1975) and Suite (‘I trl. (1975) suggcstcd that the bovine and human aortas contained acidic glycoproteins with not only .Y-glycosidic linkage, but also O-glycosidic linkage as the glycopeptide bonds. We reported here the Isolation of a glycopeptide from porcine aortas. This glycopeptide differed from those reported by the foregoing authors in the chemical composition. The present glycopeptide contained nrrther sialic acid nor sulfate. In spite of exhaustive digestron wrth pronaqe. the present glycopeptide still retained a large molecular size (about 1.200.000). The results of the [I-elimination reaction indicated that the glycopeptide had the glycosidic linkages hetwecn the N-acetylgalactosamine residues and hydroxyl grottp\ of seryl and threonyl residues. ,411 these data arc consistent with the suggestion that the present glycopeptide is derived from a mucin-type glycoprotern. It is well known that blood group activity is often associated with mucin-type glycoproteins such as submaxillary glycoprotems. gastric mucin. and ovarian cyst glycoproteins (Horowitz, 1977; Pigman. 1977a). The presence of blood group A and H

H

100re\due\

Residues

acid

AIKAWA

detectable

respectively. Fraction B exhibited no blood group B. Le”. and Leh activities at I mg/ml. DISCUSSION

Many investigators have reported glycopeptides and glycoproteins derived from aortic walls (Barnes and Partridge, 1968; Saito and Yosizawa, 1975; Saito et al., 1975; Roberts and Wagh, 1976; Heifetz et ul.,

,**.*.-“I-.

..A...-**.

0

20

Fraction

100

80

60

40

number

Fig. 6. Sephadex G-100 column chromatography of fraction B before (0) and after (0) the fi-elimination reaction. Sample was applied onto a column (I .O x I53 cm) of Sephadex G-100 preequilibrated with 0. I M ammonium bicarbonate. Elution was performed with the same solution. Fractions of 2 ml were monitored for the content of hexose. V,, and V,. see Fig. I.

Table

3 Changes

in the composltmn hexosammitol

upon

of specified

ammo

the /l-elimmation

acids.

I(-Elimination Before

Constituent Amino

hexocamme.

reaction reaction After

acid”

Aspartic Threonine

acid

Serine Glutamic Glycine

acid

Alanme

I .oo

I .(I0

9.64

3.48

4 48

3.76

4.52 2 06

4.bl 2 01

0.01

%-Aminobutyrate He\-osamme

und

1.30

ND*

5 31

He\-osaminr~ol’ I 5’) 206

Glucosamine Galactoramme

lb? II3

Glucosamimtol

ND

ND

Galactosaminitol

ND

106

“Expressed “Not

as molar

ratm

to aspartlc

detected.

‘Expressed

as @g mg

glycopeptide

acid

and

4F

A

z-

E "

0-

1

1

2

2

-21.

Fig. 4. Cellulose acetate membrane electrophoresis of fraction B in pyridine-formate buffer, pH 3.1 (A) or in the barbital buffer, pH 8.6 (B). After electrophoresis, the substances were stained by the periodic acid-Schiff reaction. 1, authentic glycogen; 2, fraction B.

Fig. 5. SDS-3% polyac~lamide (A) and SDS-l% agarose (B) gel electrophoresis of fraction B. The anode is at the bottom and the bands were visualized by the periodic acid-Schiff reaction. 1 and 3, fraction B (25~8); 2 and 4, fraction B (25 pg) reduced with 2-mercaptoethanol.

1159

Blood

group

II61

active glycopeptide

in the present glycopeptide further supports the concept that the porcine aorta contains a mucintype glycoprotein. Neiderhiser et al. (1971) reported the fucomucintype glycoproteins, which are analogous to the present glycopeptide, from the pronase digest of pig gallbladder bile. These glycoproteins also exhibited blood group A and H activities. In contrast to the gallbladder which has secretory glands. arterial tissues are solid viscera, and have no exocrine glands or secretory structures. Since most of mucin-type glycoproteins have been isolated from the tissues having mucin-secreting cells (Pigman, 1977b), the present result is rather unexpected. Thus, the present study provides the first evidence that the intima-media layer of the aortic wall contains blood group antigen(s) of mucin-type glycoprotein nature. It should be interesting to investigate the histological localization and biological function of aortic glycoprotein from which the present glycopeptide is derived.

activities

Ackno~ledgrmm/-This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education. Science and Culture of Japan.

Fukuda M., Koeffier H. P. and Minowada J. (IY81) Membrane differentiation in human myeloid cells: Expression of unique profiles of cell surface glycoproteins m myeloid leukemic cell lines blocked at different stages of differentiation and maturation. Proc,. m//11. .4c,rrt/. SC,;. U.S.A. 78, 6299-6303. Heifetr A. and Allen D. (1982) Biosynthesis of cell surface sulfated glycoproteins by cultured vascular endothclial cells. Bioc~hemi.ttr~~ 21, I7 I-- 177. Heifetz A.. Johnson A. R. and Roberts M. I(. (19X4) Synthesis of lactosaminoglycan-containing glycoproteinh by vascular endothelial cells. Biwhim. hiopll~,\. ,4(,/o 798, I -7. Holden K. G.. Yim N. C. F.. Griggs L. J. and Websbach J. A. (1971) Gel electrophoresis of mucous glycoprotclns. 1. Effect of gel porosity. Biochrmivtr~~ 10, 3105-3 109. Horowitz M. I. (1977) Gastrointestinal glycoprotclns. In The G/ycoconjugutr.s (Edited by Horowitz M. I. and Pigman W.). Vol. I, pp. 189-213. Academic Press. New York. lsemura M.. Sato N., Kikuchi M.. Munakata H.. Ototani N.. Goto N. and Yosizawa Z. (1983) Sialoglycopcptldes obtained from a transplantable rat colorectal adenocarcinoma. A comparison with those from normal colomc mucosa. Gunn 74, 373-381. Moczar M., Phan-Dinh-Tuy B.. Moczar E. and Robert L. (1983) Structural glycoproteins from rabbit aortic media. Biochem.

J. 211, 257-265.

Munakata H. and Yosizawa Z. (1980) Isolation and characterization of sulfated glycoproteins from the brush border fraction and the soluble fraction of rabbit small intestine. J. Biochem.

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Biochem.

89, 55G560.

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12.

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J. 1, 9-15.

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13262- 13267.

Dubois M., Gilles K. A.. Hamilton J. K., Rebers P. A. and Smith F. (1956) Calorimetric method for determination of sugars and related substances. Anu/]‘t. Chem. 28, 350-356.

Endo M. and Yosizawa Z. (1973) glycoproteins Archs

Biochem.

and

glycosaminoglycans

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156, 397-403.

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Neiderhiser D. H.. Plantner J. J. and Carlson D. X4. (1971) The purification and properties of the glycoproteins of pig gallbladder bile. Archs Biochem. Biophxs. 145, 155-163. Pigman W. (1977a) Blood group glycoproteins. In The Glycocorzjungatrs (Edited by Horowitz M. I. and Plgman W.), Vol. I, pp. 181-198. Academic Press, New York. Pigman W. (1977b) General aspects. In The G/~,coc,onjlrRtrtes (Edited by Horowitz M. I. and Pigman W.). Vol. 1. pp. l-1 I. Academic Press, New York. Roberts B. I. and Wagh P. V. (1976) Further studies on a highly purified glycoprotein from the intimal region of porcine aorta. Biochim. hiophyx Acts 439, 2&37. Saito H. and Yosizawa Z. (1075) Slaloglycopepttdcs isolated from bovine aorta. J. Bioc,/wm. 77, 9 I9 930. Saito H.. Ototani N. and Yosizawa Z. (1Y75) Sialoglycopeptides isolated from human artcriorclcrotic tissue. J. Biochem.

77, 931 93X.

Spiro R. G. (1972) Study of the carbohydrates of glycoproteins. In MctImI.~in En~ww/o,~~~ (Edited by Ginsburg V.). Vol. 83, pp. 269 277. Academic Prcsh. Ncu York. Tanaka K. and Pigman W. (1965) Improvements m hydrogenation procedure for demonstration of O-threonlne glycosidic linkages in bovine submaxillary mucin. J. hrol. Chem. 240, 1487 1488. Warren L. (1959) The thiobarbituric acid assay of sialic acids. J. hiol. Chem. 234, 1971-1975. Weber K. and Osborn M. (I 969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. hiol. Chem. 244, 440&4412.