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Demonstration of the existence of a specific blood-group P1 antigenic determinant in turtle-dove ovomucoid
Blood Transfusion and Immunohaematology Tome XXIII. - - N O 5. -- 1980
579
Demonstration of the existence of a specific blood-group P1 antigenic determinant in turtle-dove ovomucoid b y Ch. F R A N C O I S - G E R A R D *, J. B R O C T E U R *, A. A N D R E *, C. G E R D A Y **, A. P I E R C E - C R E T E L ***, J. M O N T R E U I L *** and G. S P I K * * *
* Laboratoire des Groupes Sanguins et de Transfusion. Universit4 de Li6ge, 4020 LIEGE. ** Laboratoire de Biochimie Musculaire, Universit4 de Lihge, 4000 LIEGE, *** Laboratoire de Chimie Biologique, Universit6 des Sciences et Techniques de Lille I, VILLENEUVE-D'Asco(Nord).
INTRODUCTION
TURTLE-DOVE egg-white presents a very strong P1 antigenic activity carried by a glycoprotein called ovomucoid. This P~ cross-reacting material can be c o m p a r e d with the hydatid cyst glycoprotein [1, 2 ] : both inhibit anti-P~ and anti-P k antibodies [3] and are also able to induce agglutinating anti-P~ when injected to goats and rabbits [4]. Ovomucoid f r o m turtle-dove egg-white is h o m o g e n e o u s in size b u t heterogeneous in charge. It consists, in fact, in at least five components differing mainly by their NeuAc residues contents. We previously purified o v o m u c o i d f r o m turtle-dove egg-white and characterized some of its physico-chemical and biological properties [5]. In transfusional practice, turtle-dove ovomucoid is c o m m o n l y used in the Blood Group L a b o r a t o r y of Libge as specific anti-P~ inhibitor, especially in the cases of polyimmunisations when an anti-P 1 antibody is suspected to be involved in a mixure of cold agglutinins [6]. The present data deal with the s t r u c t u r e determination of one of
580
FRANCOIS-GERARD
Ch. and coll.
the glycopeptides obtained by enzymic proteolysis of total ovomucoid. By reference to the h u m a n e r y t h r o c y t e P1 antigen whose structure has been elucidated by NAIKI et al. [7], we looked for the presence of a terminal trisaccharide c o r r e s p o n d i n g to the s e q u e n c e : ~-Gal(1 ~ 4)-fi-Gal-(1 ~ 4)-fi-GlcNAc. As expected, such a s t r u c t u r e has been f o u n d in turtle-dove ovomucoid.
MATERIAL AND METHODS Turtle-dove ovomucoid purification. Ovomucoid is extracted f r o m turtle-dove egg-white (Streptopelia Risoria) by trichloracetic acid-acetone precipitation according to Lineweaver and M u r r a y [8]. F u r t h e r purification is p e r f o r m e d by ionexchange c h r o m a t o g r a p h y on DEAE-cellulose in the experimental conditions described by FEENEY et al. [9]. The purity of o v o m u c o i d is tested by isoelectrofocusing in polyacrylamide gels as previously described [5].
Glycopeptides preparation and purification. Pure o v o m u c o i d previously c a r b o x y m e t h y l a t e d [10] is incubated for 15 h. at 37°C. with 2 % (w/w) of p r o n a s e (Calbiochem N ° 537 088 free of nucleases, grade B) in 50 ml. of b o r a t e buffer pH 8 (Na2B407 0.025 M containing CaC] 2 0.01 M). On a c c o u n t of their size, the glycopeptides are isolated f r o m the p r o d u c t s of digestion by gel filtration on a Biogel P2 c o l u m n (140 × 2.5 cm) equilibrated in distilled water. The mixture of glycopeptides is f u r t h e r separated into six m a j o r peaks by ion-exchange c h r o m a t o g r a p h y on a P-cellulose c o l u m n (26 × 2 cm) equilibrated in 0.02 N H~PO4 p H 2.45. The c o l u m n is first eluted with the buffer (150 ml.) and then with a gradient of ionic strength ( 0 - - 0 . 2 M KC1, 200/200 ml.). The m o s t basic c o m p o n e n t , called Pr5, is f u r t h e r purified by c h r o m a t o g r a p h y on DEAE-cellulose (15 × 1.5 cm) equilibrated in a 0.015 M HC1-Tris b u f f e r p H 8. Elution of the peptides requires an increase of ionic strength f r o m 0 to 0.3 M NaC1 (150/150 ml.).
Structure analysis. ANALYTICAL. Hexoses content has been d e t e r m i n e d by the phenol-sulfuric m e t h o d of DUBOIS et al. [11] using an equimolar mixture of m a n n o s e and galactose as standard. Individual m o n o s a c c h a r i d e s and hexosamines were assayed by g.l.c. after methanolysis of the samples according to ZANETTA et al. [12] and using mesoinositol as internal standard. Amino acid analyses were carried out after hydrolysis with 6 M HC1 for 24 h. at 110° C. in evacuated sealed tubes, following the m e t h o d of BENSON and PATTERSON [13] and using ~ B e c k m a n Model 120 B Amino Acid Analyser.
581
SPECIFIC BLOOD-GROUP P1
METHYLATION.
The basic glycopeptide Pr5 isolated and purified by ion-exchange c h r o m a t o g r a p h y has b e e n p e r m e t h y l a t e d according to the m e t h o d of HAKOMORI [14]. H e x o s a m i n e derivatives w e r e m e a s u r e d a f t e r acetylation of the m e t h y l a t e d p r o d u c t s by addition of 200 ~1 of pyridine and 200 Ixl of acetic a n h y d r i d e for 45 m i n u t e s at 150°C [15]. The m e t h y l a t e d products w e r e identified by g.l.c, a n d f u r t h e r analyzed by m a s s spectrom e t r y (Riber-Mag 10-10; Rucil-Malmaison, France). HYDRAZINOLYSIS.
A s a m p l e of glycopeptide Pr5 (2 mg) has been s u b m i t t e d to hydrazinolysis followed by diazotation as described b y BAYARD and MONTREUIL [16]. The resulting f r a g m e n t s w e r e identified by m a s s spectrom e t r y according to the m e t h o d u s e d by STRECKER et al. [17]. ENZYMIC HYDROLYSIS OF GLYCOPEPTIOE Pv5 :
¢-galactosidase (EC.3.2.1.22) was o b t a i n e d f r o m Aspergillus Niger [ 18 ]. The enzymic activity of the p r e p a r a t i o n was e s t i m a t e d by hydrolysis of para-nitro-phenyl-c~-D-galacto-pyrannoside dissolved in a 0.1 M phosphate-citrate b u f f e r p H 3.5. Purified Pr5 glycopeptide (2 mg.) dissolved in 500 ~1 of b u f f e r ( p H 3.5 containing 1%0 toluol) was incubated with 2 rag. of enzyme for 120 h. at 37°C. Aliquots of 50 ~1 of the digestion m i x t u r e w e r e t a k e n at various times of hydrolysis and analyzed for PI antigenic activity. MEASUREMENT OF P1 ACTIVITY:
H u m a n e r y t h r o c y t e s w e r e o b t a i n e d f r o m blood donors belonging to the PI strong phenotype. Goat anti-P 1 s e r u m was a gift f r o m Professor O. P r o k o p (Berlin, E. Germany). W a s h e d e r y t h r o c y t e s suspended in iso-osmotic b u f f e r I s o t o n I I (Coulter Electronics) w e r e i n c u b a t e d for 2 h. in the p r e s e n c e of decreasing concentrations of the Pl-active Pr5 glycopeptide. Residual h a e m a g g l u t i n a t i o n was m e a s u r e d by using a Coulter Model B a u t o m a t i c cell Counter. The p e r c e n t a g e of agglutinated red cells s u b m i t t e d to the logit t r a n s f o r m a t i o n [19] was plotted on a double logarithmic scale as a function of the inhibitor concentration. This t r e a t m e n t allowed us to calculate, for each sample, the concentration responsible for 50 % of inhibition, and consequently, the relative loss of Pl-like activity during the enzymic digestion.
RESULTS Turtle-dove o v o m u c o i d purification. The purification of turtle-dove o v o m u c o i d gives rise to a p u r e protein p r e p a r a t i o n , containing five c o m p o n e n t s , as was s h o w n by isoelectrofocusing in p o l y a c r y l a m i d e gel. S o m e physico-chemical
582
FRANCOIS-GERARD
Ch. and coll.
c h a r a c t e r i s t i c s of t u r t l e - d o v e o v o m u c o i d h a v e p r e v i o u s l y b e e n r e p o r t e d as w e l l as its t o t a l a m i n o acid c o m p o s i t i o n [5]. Like m o s t glycoproteins, t h e c a r b o h y d r a t e m o i e t y of t u r t l e - d o v e o v o m u c o i d c o n t a i n s : m a n n o s e , galactose, N - a c e t y l - g l u c o s a m i n e a n d N - a c e t y l - n e u r a m i n i c acid. T h e i r r e l a t i v e a m o u n t , e x p r e s s e d as t h e n u m b e r of r e s i d u e s p e r m o l e of t u r t l e - d o v e o v o m u c o i d (29400 M.W.) is r e s p e c t i v e l y : GAL (17), MAR (9), GlcNAc (19) a n d N e u A c (0-2). The NeuAc c o n t e n t was s h o w n to v a r y f r o m o n e c o m p o n e n t to t h e o t h e r [4].
Glycopeptide preparation and purification. T h e o v o m u c o i d s u b m i t t e d to p r o n a s i c d i g e s t i o n w a s p r e v i o u s l y c a r b o x y m e t h y l a t e d i n o r d e r to i n c r e a s e t h e s p e e d of h y d r o l y s i s w h i c h a p p e a r s , i n this case, to o c c u r l0 t i m e s f a s t e r [20]. T h e c h e m i c a l m o d i f i c a t i o n of t h e g l y c o p r o t e i n does n o t i m p a i r its P~-like activity.
A 215 nn 489nm
~z~ m
mho
20
A
lo
50
100
150
200
fraclions
FIG_ 1. - - Chromatography of the mixture of glycopeptides on a P-cellulose column (26 × 2 cm) equilibrated in 0.02 N H~PO4 pH 2.45. Elution is performed by applying a gradient of ionic strength: 1° 150 ml. 0.02 N HaPO4, 2° 200/200 ml. 0.02 N HaPO4 containing O - 0.2 M KCI. The absorbancy of each fraction (3 ml.) is measured at 215 nm (e--o) and at 489 nm ( / k - - - A ) after a phenol-sulfuric reaction performed with 200 Ial aliquots. The increase of ionic strength is illustrated by the conductivity of the eluate ( 0 - - 0 ) . T h e u n r e t a r d e d p e a k o b t a i n e d o n t h e Biogel-P a c o l u m n c o n t a i n s all t h e g l y c o p e p t i d e s as s h o w n b y t h e p h e n o l - s u l f u r i c test. These f r a c t i o n s w e r e p o o l e d , l y o p h i l y z e d a n d s u b m i t t e d to 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 n a P-cellulose c o l u m n . T h e e l u t i o n p r o f i l e is s h o w n i n Fig. 1. O u t of six m a j o r p e a k s , o n l y o n e is free of a n y glycosidic
SPECIFIC BLOOD-GROUP Pt
583
r e s i d u e ; the others have been n a m e d P r l to Pr5 in the o r d e r of elution. The m o s t basic one, PrS, is also the m o s t active anti-P I inhibitor. T h a t is the r e a s o n w h y we have, at first, u n d e r t a k e n the study of the Pl-like antigenic s t r u c t u r e on this glycopeptide. The glycopeptide was f u r t h e r purified b y c h r o m a t o g r a p h y on DEAEcellulose (Fig. 2). I t a p p e a r s to be p u r e as checked by p a p e r electrophoresis at p H 2.4 in 1 M acetic acid.
FIG.
2. - - C h r o m a t o g r a p h y of Pr5 glycopeptide o n a DEAE-cellulose c o l u m n (15 × 1.5 cm) e q u i l i b r a t e d in a Tris-HC1 pH 8 buffer (0.015 M HC1 Tris). E l u t i o n is e n s u r e d by a g r a d i e n t of ionic s t r e n g t h f r o m 0 to 0.3 M NaCI in a m i x t u r e of 150/150 m l of b u f f e r . The a b s o r b a n c y of each f r a c t i o n (2 ml.) is m e a s u r e d at 215 n m (e--e). The p h e n o l - s u l f u r i c reaction a p p l i e d to 200 ~1. of a l i q u o t is r e a d at 489 n m ( ~ - - ' A ) . The c o n d u c t i v i t y of the eluate s h o w s the slope of the ionic s t r e n g t h increase ( Q - - G).
I
50
10D rraclions
Structure analysis of Pr5. AMINO ACID AND SUGAR COMPOSITION.
The osidic c o m p o s i t i o n of glycopeptide Pr5 d e t e r m i n e d by methanolysis is s u m m a r i z e d in Table I which also presents the a m i n o acid content of the peptidic moiety. TABLE I
A m i n o acid composition and carbohydrate content of glycopeptide Pr5 expressed as n u m b e r of residues per mole of glycopeptide.
AMIN0 ACID : His . . . . . . . . . . . . . . . . . . . . . . . . . ASX . . . . . . . . . . . . . . . . . . . . . . . . . . Pro . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1 1 0.5
(1) (1) (o-D
5.7 3 6.5
(6) (3) (7-8)
CARBOHYDRATE : Gal . . . . . . . . . . . . . . . . . . . . . . . . . . Man . . . . . . . . . . . . . . . . . . . . . . . . GIcNAc . . . . . . . . . . . . . . . . . . . . . .
584
FRANCOIS-GERARD Ch. and coll.
METHYLATION. T h e a n a l y s i s o f t h e m e t h y l a t e d d e r i v a t i v e s of Pr5 w a s c o n s i s t e n t w i t h g a l a c t o s e r e s i d u e s i n t e r m i n a l p o s i t i o n s i n c e a l a r g e a m o u n t of m e t h y l 2,3,4,6-tetra-O-methyl g a l a c t o s i d e c o u l d b e i d e n t i f i e d b y g.l.c. T h e p r e s e n c e o f b o t h m e t h y l 2,3,6,-tri-O-methyl g a l a c t o s i d e a n d m e t h y l 3,6-di-O-methyl-N-(acetyl) m e t h y l g l u c o s a m i n i d e d e n o t e s t h a t G a l a n d G l c N A c r e s i d u e s a r e s u b s t i t u t e d in t h e C 4 p o s i t i o n . T a b l e I I s u m m a r i z e s all t h e m e t h y l a t e d d e r i v a t i v e s t h a t c o u l d b e i d e n t i f i e d b y g.l.c. TABLE II Identification by g.l.c, and molar ratio of the O-methyl glycosides present in the Pr5 glycopeptide after methylation.
2, 2, 3, 2, 3 2 2, '3,
METHYLATEDMONOSACCHARIDES
MOLAR RATIO
3, 4, 6 - - tetra-O-methyl-galactoside . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 - - tri-O-methyl-galactoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 - - di-O-methyl rnannoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 - - di-O-methyl malanoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - mono-O-methyl mannoside . . . . . . . . . . . . . . . . . . . . . . . . . . - - InOlao-O-methyl mannoside . . . . . . . . . . . . . . . . . . . . . . . . . . 3, 4, 6 - - tetra-O-N-acetyl-rnethyl glucosaminide . . . . . . . . . . . . . . 6 - - di-O-acetyl-methyl glucosaminide . . . . . . . . . . . . . . . . . . . .
+ 3 1 1 1 1
+ 5
HYDRAZINOLYSIS. Hydrazinolysis followed by nitrous deamination specifically cleaves t h e G l c N A c 0 - g l y c o s i d i c l i n k a g e s , c o n v e r t i n g G l c N A c i n t o 2,5-anhydrom a n n o s e . W h e n t h i s l a t t e r p r o d u c t is r e d u c e d , it is i d e n t i f i e d as 2,5a n h y d r o m a n n i t o l in g.l.c. I n t h e p r o d u c t s l i b e r a t e d b y t h i s m e t h o d , a t r i s a c c h a r i d e w a s f i r s t s e p a r a t e d b y g.l.c, a n d t h e n i d e n t i f i e d b y m a s s s p e c t r o m e t r y as c o r r e s p o n d i n g to t h e s e q u e n c e : Gal-Gal-2,5anhydromannitol. ENZYMIC DIGESTION AND IOa-LIKE ACTIVITY. T h e ~ - g a l a c t o s i d a s e i s o l a t e d f r o m A s p e r g i l l u s N i g e r w a s p r o v e d to b e f r e e of a n y o t h e r g l y c o s i d a s i c a c t i v i t y b y t e s t i n g s e v e r a l s y n t h e t i c s u b s t r a t e s . I t s a c t i v i t y is e q u i v a l e n t to 0.2 ~ m o l e of p a r a - n i t r o - p h e n y l a - D - g a l a c t o p y r a n n o s i d e h y d r o l y z e d a t 37°C. p e r m i n u t e a n d p e r rag. of protein. A s i g n i f i c a n t loss o f P~-activity w a s o b s e r v e d a f t e r 120 h. of d i g e s t i o n u s i n g a 1/1 ( w / w ) e n z y m e t o p e p t i d e r a t i o . Fig. 3 s h o w s t h e e v o l u t i o n of t h e PI a c t i v i t y as a f u n c t i o n of t i m e . T h e p e r c e n t a g e of r e m a i n i n g a c t i v i t y , e x p r e s s e d as t h e c o n c e n t r a t i o n r e s p o n s i b l e f o r 50 % of h a e m a g g l u t i n a t i o n i n h i b i t i o n , d e c r e a s e s f o l l o w i n g a n e x p o n e n t i a l curve. T h e a n a l y s i s a f t e r m e t h a n o l y s i s of t h e p r o d u c t of d i g e s t i o n r e v e a l s a s i g n i f i c a n t l o s s of t h r e e g a l a c t o s e r e s i d u e s . F u r t h e r m o r e , m e t h y l a t i o n
SPECIFIC BLOOD-GROUP P1
585
of the e-galactosidase t r e a t e d glycopeptide showed a decrease of m e t h y l 2,3,6-tri-0-methyl galactoside whereas the a m o u n t of m e t h y l 2,3,4,6-tetra0-methyl galactoside r e m a i n s identical. This suggests the p r e s e n c e of Gal residues at the non reducing position, linked to o t h e r Gal residues by a ~-1,4 type linkages.
!' 0'5
FIG. 3, - - P1 activity oE g l y c o p e p t i d e Pr5 d i g e s t e d w i t h c~-ga|actosidase. T h e r e s u l t s a r e e x p r e s s e d as t h e fractional activity giving 50 % inhibition of haemagglu tination
0
(C 50 %).
I
l 20
40
GO
80
100
lime
(hou,s~
DISCUSSION
The N-glycosidically linked glycans of m o s t glycoproteins are built following a classical p e n t a s a c c h a r i d i c s t r u c t u r e involving a b r a n c h e d m a n n o s i d i c core, m a d e of three residues, linked to the peptidic chain by two sequential GlcNAc residues [21]. The two external m a n n o s e residues are p r e f e r e n t i a l sites for attaching two glycosidic chains. The possibility of m o r e b r a n c h e d glycannic s t r u c t u r e s cannot be, however, ruled out [22]. As turtle-dove o v o m u c o i d glycopeptide Pr5 is concerned, the presence of mono-, di- and tri-0-methyl m a n n o s i d e in the m e t h y l a t e d derivatives of Pr5, is in favour of a p o l y b r a n c h e d structure. Taking into account the P1 antigenic s t r u c t u r e s of b o t h h u m a n glycosphingolipid [7] and hydatid cyst glycoprotein [23], w e first looked for ~-Gal residues in t e r m i n a l position by using b o t h chemical and enzymatic m e t h o d s . The p r e s e n c e of tetra-0-methyl galactoside in the m e t h y l a t e d p r o d u c t s and the loss of P1 activity during ~-galactosidase digestion c o n f i r m s t i m u l t a n e o u s l y the existence of g-galactose at the non reducing end of the glycosidic chain. It m a y t h e r e f o r e be stated that ~-Gal is the i m m u n o d o m i n a n t sugar of the P~-like antigenic d e t e r m i n a n t of turtle-dove ovomucoid, as in the case of h u m a n erythrocyte and h y d a t i d antigen. Moreover, the lost of three Gal residues after exhaustive e-galactosidase t r e a t m e n t and the p r e s e n c e of a single p e a k c o r r e s p o n d i n g to the trisaccharide Gal-Gal-2,5 a n h y d r o m a n n i t o l , identified by m a s s s p e c t r o m e t r y in the hydrazinolysate of native Pr5 suggest t h a t three Pl-active trisaccharides are p r o b a b l y p r e s e n t per m o l e of glycopeptide.
586
FRANCOIS-GERARD
Ch. and coll.
Pooling all results o b t a i n e d b y chemical and enzymatical ways, we p r o p o s e the following s t r u c t u r e (Fig. 4) for the Pl-like antigenic d e t e r m i n a n t associated to the Pr5 glycopeptide of turtle-dove ovomucoid. I t w o u l d b e h a z a r d o u s to establish a definite s t r u c t u r e for the glycopeptide Pr5 as long as the o t h e r glycopeptidic s t r u c t u r e s of turtle-dove o v o m u c o i d are not yet c o m p l e t e l y elucidated. According to the analyses u n d e r t a k e n to c h a r a c t e r i z e the o t h e r glycopeptides, we t h i n k t h a t t h r e e different glycosylation sites t a k e place on the polypeptide chain of turtle-dove ovomucoid, each of t h e m p r o d u c i n g a Pl-active glycopeptide.
GIENAc
(1-4) GIcNAc (1--2)
Man
GIcNAc (1~4) Man (I~4)GIcNAc (x--4)G[cNAc ~ A s n
/-6,a) ¢=Ga[ (1-4) Gal (1-4) GIcNAc (1~2) Man GIcNAc /~4) GBI /--4) ~-Gal
~/(~-o)
(1-3)
G[cNAc I (1--4) Ga[
I,1-4)
-Gal
FIG. 4. - - H y p o t h e t i c a l s t r u c t u r e p r o p o s e d f o r o n e of t h e P1active g l y c o p e p t i d e s i s o l a t e d f r o m t u r t l e - d o v e o v o m u e o i d b y p r o n a s i c digestion.
RESUME L ' o v o m u c o i d e pr6sent dans le blanc d'.ceuf de tourterelle est une nouvelle s u b s t a n c e soluble d o n n a n t lieu ~ une rdaction crois4e avec l'antighne P~ humain. L ' o v o m u c o i d e est une glycoprot6ine de P.M. 29 400, c o n t e n a n t envir o n 20 % de rdsidus osidiques r6partis e n : m a n n o s e , galactose, NacEtyl glucosamine et acide N-acdtyl-neuraminique. C o m m e la glycoprot6ine du liquide de kyste hydatique, l'ovomucoide de tourterelle est un i m m u n o g h n e efficace p u i s q u e d'excellents s6rums anti-P 1 ont 6t4 obtenus p a r i m m u n i s a t i o n de lapins. P o u r c o n n a i t r e la s4quence osidique s p d c i f i q u e m e n t r e s p o n s a b l e de l'activit6 antig6nique P1, l ' o v o m u c o i d e de tourterelle a 6t6 fragmental par digestion pronasique. Les glycopeptides Pl-actifs ont 6t6 sdpar6s par filtration mol4culaire puis purifies p a r c h r o m a t o g r a p h i e sur 6changeurs d'ions. Le plus actif d ' e n t r e eux, le f r a g m e n t Pr 5, a 6t6 l'objet d'une r e c h e r c h e plus approfondie.
SPECIFIC BLOOD-GROUP PI
587
Les m 6 t h o d e s p h y s i c o - c h i m i q u e s telles la c h r o m a t o g r a p h i e e n p h a s e gazeuse et la s p e c t r o m 6 t r i e de m a s s e p e r m e t t e n t de c o n c l u r e q u ' u n e s 6 q u e n c e t r i s a c c h a r i d i q u e ~-Gal 1 ~ 4 ~-Gal 1 ~ 4 ~ GlcNAc i d e n t i q u e /~ celle q u i c a r a c t 6 r i s e l ' a n t i g 6 n e P~ 6 r y t h r o c y t a i r e , est e f f e c t i v e m e n t pr6s e n t e d a n s l ' o v o m u c o i d e de t o u r t e r e l l e . De p l u s , la d i g e s t i o n e n z y m a t i q u e p a r l ' ¢ - g a l a c t o s i d a s e , q u i e n t r a i n e u n e p e r t e de l ' a c t i v i t 6 P~-like, conf i r m e q u ' u n r 6 s i d u d'~-Gal est 6 g a l e m e n t le s u c r e i m m u n o d o m i n a n t d u d d t e r m i n a n t P1 de l ' o v o m u c o i d e . Les r e c h e r c h e s e n t r e p r i s e s p o u r c a r a c t 6 r i s e r les a u t r e s f r a g m e n t s Pl-actifs de l ' o v o m u c o i d e , a m 6 n e n t ~ p e n s e r q u ' i l existe 3 sites de glycos y l a t i o n p a r m o l 6 c u l e d ' o v o m u c o i d e , c h a c u n d ' e u x p r o d u i s a n t u n glycop e p t i d e P~-actif.
Request reprints f r o m : Ch. FRAN§OIS-GF;RARD, Universit~ de Li6ge, Laboratoire des Groupes Sanguins et de Transfusion, 41, rue Dos-Fanchon, B-4020 LI~?GE (Belgique).
REFERENCES [1] CAMERONG.L. a n d STAVELEY J.M. - - Blood group P substance in hydatid cyst fluids. Nature London, 179, 147 (1957). [2] CORY H.T., YATES A.D., DONALDA.S.R., WATKINS W.M. and MORGAN W.T.J. - - The n a t u r e of the h u m a n blood group P1 determinant. Bioehem. Biophys. Res. Comm., 61, 1289 (1974). [3] VOAKD., ANSTEE D. and PARDOEG. - - The ~-galactose specificity of anti-P~. Vox Sang., 25, 263 (1973). [4] FRANoCOIS-Gt~RARDCh., BROCTEUR J. and ANDRI~ A. - - Turtle-dove: a n e w source of Pl-like m a t e r i a l cross-reacting with the h u m a n erythrocyte antigen. Fox Sang. (in press). [5] FRANCOIs-GI~,RARDCh., GERDAY C. and BEELEY J.G. - - Turtle-dove ovomuc o i d ; a glycoprotein proteinase inhibitor with Pl-blood group antigen activity. Biochem. J., 277, 679 (1979). [6] FRAN,COIs-GP-RARDCh. et BROCTEOR J. - - Application /~ la s6rologie transfusionnelle du caractbre antig6nique Pl do blanc d',ceuf de pigeon. Rev. Franf. Transl. et Immunohdmatol. (in press). [7] NAIKI M., FONG J., LEDEEN R. a n d MARCUS D.M. - - Structure of the h u m a n erythrocyte blood group P1 glycosphingolipid. Biochemistry, 14, 4831 (1975). [8] LI~EWEAVERH. and MURRAY C.W. - - Identification of the trypsin inhibitor of egg-whit ovomucoid. J. Biol. Chem., 171, 565 (1947). [9] FEENEY R.E., OSUGAD.T. and MAEDA H. - - Heterogeneity of avian ovomucolds. Arch. Biochem. Biophys., 119, 124 (1967). [10] CRETSFIELD A.M., MOORE S. and STEIN W.H. - - The p r e p a r a t i o n and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J_ Biol. Chem., 238, 622 (1963).
588
FRANCOIS-GERARD
Ch. and coll.
[11] DUBOlS M., GILLES K.A., HAMILTON J.K., REBERS P.A_ and SMITH F. - Colorimetric m e t h o d for d e t e r m i n a t i o n of sugars and released substances. Anal. Chem., 28, 350 (1956). [12] ZANETTA J.P., BRECKENRIDGE W . C . a n d VINCENTON G. - - Analysis of monosaccharides by g.l.c, of the O-methylglycosides as trifluoroacetate derivatives. Application to glycoproteins a n d glycolipids. J. Chromatogr., 69, 291 (1872). [13] BENSON J.V. a n d PATTERSONJ'.A. m Accelerated automatic chromatographic analysis of a m i n o acids on a spherical resin. Anal. Chem., 37, 1108 (1965). [14] HAKOMORIS.I. - - A rapid p e r m e t h y l a t i o n of glycolipid a n d polysaccharide catalyzed b y methyl sulfinyl c a r b a n i o n in dimetyl sulfoxide_ Y. Biochem., 55, 205 (1964). [15] FOURNET B. - - (Personal communication). [16] BAYARD B. a n d MONTREUIL J. - - Specific cleavage of N-acetyl glucosam i n i d i c linkages. Colloques Internationaux du C.N.R.S., 221, 209 (1973). [17] STRECKER G., FOURNET B., MONTREUIL ]'., DORLAND L., HAVERKAMP J. a n d VLIEGENTHART J.F.G. - - S t r u c t u r e of the three m a j o r fucosyl-glycoaspargines a c c u m u l a t i n g in the u r i n e of a p a t i e n t with fucosidosis. Biochimie, 60, 725 (1978). [18] BOUOUELET S., SPIK G. and MONTREUIL ft. - - Properties of a a-mannosidase from Aspergillus Niger. Biochim. Biophys. Acta, 522, 521 (1978). [19] ASTHON W.D. - - The logit t r a n s f o r m a t i o n with special reference to its uses in bioassay. Griffin's Statistical Monographs a n d Courses (Ed. Alan Stuart, 1972). [20] FRAN§OIs-GI~RARDCh. - - (Unpublished results). [21] MONTREUILJ. - - Recent data on the s t r u c t u r e of the c a r b o h y d r a t e moiety of glycoproteins. Metabolism and biological implications. Pure and applied Chemistry, 42, 431 (1975). [22] STRECKER G. and MONTREUIL J. - - Glycoproteins and glycoproteinoses. Biochimie, 61, 1199 (1979). [23] WATKINS W.M. a n d MORGAN W.T.J. - - Blood group P1 s u b s t a n c e : II I m m u n o l o g i c a l properties. Proc. 9th Congr. int. Soc. Blood Transf. Mexico, 1962. S. Karger B~tle 230 (1964).
579
Demonstration of the existence of a specific blood-group P1 antigenic determinant in turtle-dove ovomucoid b y Ch. F R A N C O I S - G E R A R D *, J. B R O C T E U R *, A. A N D R E *, C. G E R D A Y **, A. P I E R C E - C R E T E L ***, J. M O N T R E U I L *** and G. S P I K * * *
* Laboratoire des Groupes Sanguins et de Transfusion. Universit4 de Li6ge, 4020 LIEGE. ** Laboratoire de Biochimie Musculaire, Universit4 de Lihge, 4000 LIEGE, *** Laboratoire de Chimie Biologique, Universit6 des Sciences et Techniques de Lille I, VILLENEUVE-D'Asco(Nord).
INTRODUCTION
TURTLE-DOVE egg-white presents a very strong P1 antigenic activity carried by a glycoprotein called ovomucoid. This P~ cross-reacting material can be c o m p a r e d with the hydatid cyst glycoprotein [1, 2 ] : both inhibit anti-P~ and anti-P k antibodies [3] and are also able to induce agglutinating anti-P~ when injected to goats and rabbits [4]. Ovomucoid f r o m turtle-dove egg-white is h o m o g e n e o u s in size b u t heterogeneous in charge. It consists, in fact, in at least five components differing mainly by their NeuAc residues contents. We previously purified o v o m u c o i d f r o m turtle-dove egg-white and characterized some of its physico-chemical and biological properties [5]. In transfusional practice, turtle-dove ovomucoid is c o m m o n l y used in the Blood Group L a b o r a t o r y of Libge as specific anti-P~ inhibitor, especially in the cases of polyimmunisations when an anti-P 1 antibody is suspected to be involved in a mixure of cold agglutinins [6]. The present data deal with the s t r u c t u r e determination of one of
580
FRANCOIS-GERARD
Ch. and coll.
the glycopeptides obtained by enzymic proteolysis of total ovomucoid. By reference to the h u m a n e r y t h r o c y t e P1 antigen whose structure has been elucidated by NAIKI et al. [7], we looked for the presence of a terminal trisaccharide c o r r e s p o n d i n g to the s e q u e n c e : ~-Gal(1 ~ 4)-fi-Gal-(1 ~ 4)-fi-GlcNAc. As expected, such a s t r u c t u r e has been f o u n d in turtle-dove ovomucoid.
MATERIAL AND METHODS Turtle-dove ovomucoid purification. Ovomucoid is extracted f r o m turtle-dove egg-white (Streptopelia Risoria) by trichloracetic acid-acetone precipitation according to Lineweaver and M u r r a y [8]. F u r t h e r purification is p e r f o r m e d by ionexchange c h r o m a t o g r a p h y on DEAE-cellulose in the experimental conditions described by FEENEY et al. [9]. The purity of o v o m u c o i d is tested by isoelectrofocusing in polyacrylamide gels as previously described [5].
Glycopeptides preparation and purification. Pure o v o m u c o i d previously c a r b o x y m e t h y l a t e d [10] is incubated for 15 h. at 37°C. with 2 % (w/w) of p r o n a s e (Calbiochem N ° 537 088 free of nucleases, grade B) in 50 ml. of b o r a t e buffer pH 8 (Na2B407 0.025 M containing CaC] 2 0.01 M). On a c c o u n t of their size, the glycopeptides are isolated f r o m the p r o d u c t s of digestion by gel filtration on a Biogel P2 c o l u m n (140 × 2.5 cm) equilibrated in distilled water. The mixture of glycopeptides is f u r t h e r separated into six m a j o r peaks by ion-exchange c h r o m a t o g r a p h y on a P-cellulose c o l u m n (26 × 2 cm) equilibrated in 0.02 N H~PO4 p H 2.45. The c o l u m n is first eluted with the buffer (150 ml.) and then with a gradient of ionic strength ( 0 - - 0 . 2 M KC1, 200/200 ml.). The m o s t basic c o m p o n e n t , called Pr5, is f u r t h e r purified by c h r o m a t o g r a p h y on DEAE-cellulose (15 × 1.5 cm) equilibrated in a 0.015 M HC1-Tris b u f f e r p H 8. Elution of the peptides requires an increase of ionic strength f r o m 0 to 0.3 M NaC1 (150/150 ml.).
Structure analysis. ANALYTICAL. Hexoses content has been d e t e r m i n e d by the phenol-sulfuric m e t h o d of DUBOIS et al. [11] using an equimolar mixture of m a n n o s e and galactose as standard. Individual m o n o s a c c h a r i d e s and hexosamines were assayed by g.l.c. after methanolysis of the samples according to ZANETTA et al. [12] and using mesoinositol as internal standard. Amino acid analyses were carried out after hydrolysis with 6 M HC1 for 24 h. at 110° C. in evacuated sealed tubes, following the m e t h o d of BENSON and PATTERSON [13] and using ~ B e c k m a n Model 120 B Amino Acid Analyser.
581
SPECIFIC BLOOD-GROUP P1
METHYLATION.
The basic glycopeptide Pr5 isolated and purified by ion-exchange c h r o m a t o g r a p h y has b e e n p e r m e t h y l a t e d according to the m e t h o d of HAKOMORI [14]. H e x o s a m i n e derivatives w e r e m e a s u r e d a f t e r acetylation of the m e t h y l a t e d p r o d u c t s by addition of 200 ~1 of pyridine and 200 Ixl of acetic a n h y d r i d e for 45 m i n u t e s at 150°C [15]. The m e t h y l a t e d products w e r e identified by g.l.c, a n d f u r t h e r analyzed by m a s s spectrom e t r y (Riber-Mag 10-10; Rucil-Malmaison, France). HYDRAZINOLYSIS.
A s a m p l e of glycopeptide Pr5 (2 mg) has been s u b m i t t e d to hydrazinolysis followed by diazotation as described b y BAYARD and MONTREUIL [16]. The resulting f r a g m e n t s w e r e identified by m a s s spectrom e t r y according to the m e t h o d u s e d by STRECKER et al. [17]. ENZYMIC HYDROLYSIS OF GLYCOPEPTIOE Pv5 :
¢-galactosidase (EC.3.2.1.22) was o b t a i n e d f r o m Aspergillus Niger [ 18 ]. The enzymic activity of the p r e p a r a t i o n was e s t i m a t e d by hydrolysis of para-nitro-phenyl-c~-D-galacto-pyrannoside dissolved in a 0.1 M phosphate-citrate b u f f e r p H 3.5. Purified Pr5 glycopeptide (2 mg.) dissolved in 500 ~1 of b u f f e r ( p H 3.5 containing 1%0 toluol) was incubated with 2 rag. of enzyme for 120 h. at 37°C. Aliquots of 50 ~1 of the digestion m i x t u r e w e r e t a k e n at various times of hydrolysis and analyzed for PI antigenic activity. MEASUREMENT OF P1 ACTIVITY:
H u m a n e r y t h r o c y t e s w e r e o b t a i n e d f r o m blood donors belonging to the PI strong phenotype. Goat anti-P 1 s e r u m was a gift f r o m Professor O. P r o k o p (Berlin, E. Germany). W a s h e d e r y t h r o c y t e s suspended in iso-osmotic b u f f e r I s o t o n I I (Coulter Electronics) w e r e i n c u b a t e d for 2 h. in the p r e s e n c e of decreasing concentrations of the Pl-active Pr5 glycopeptide. Residual h a e m a g g l u t i n a t i o n was m e a s u r e d by using a Coulter Model B a u t o m a t i c cell Counter. The p e r c e n t a g e of agglutinated red cells s u b m i t t e d to the logit t r a n s f o r m a t i o n [19] was plotted on a double logarithmic scale as a function of the inhibitor concentration. This t r e a t m e n t allowed us to calculate, for each sample, the concentration responsible for 50 % of inhibition, and consequently, the relative loss of Pl-like activity during the enzymic digestion.
RESULTS Turtle-dove o v o m u c o i d purification. The purification of turtle-dove o v o m u c o i d gives rise to a p u r e protein p r e p a r a t i o n , containing five c o m p o n e n t s , as was s h o w n by isoelectrofocusing in p o l y a c r y l a m i d e gel. S o m e physico-chemical
582
FRANCOIS-GERARD
Ch. and coll.
c h a r a c t e r i s t i c s of t u r t l e - d o v e o v o m u c o i d h a v e p r e v i o u s l y b e e n r e p o r t e d as w e l l as its t o t a l a m i n o acid c o m p o s i t i o n [5]. Like m o s t glycoproteins, t h e c a r b o h y d r a t e m o i e t y of t u r t l e - d o v e o v o m u c o i d c o n t a i n s : m a n n o s e , galactose, N - a c e t y l - g l u c o s a m i n e a n d N - a c e t y l - n e u r a m i n i c acid. T h e i r r e l a t i v e a m o u n t , e x p r e s s e d as t h e n u m b e r of r e s i d u e s p e r m o l e of t u r t l e - d o v e o v o m u c o i d (29400 M.W.) is r e s p e c t i v e l y : GAL (17), MAR (9), GlcNAc (19) a n d N e u A c (0-2). The NeuAc c o n t e n t was s h o w n to v a r y f r o m o n e c o m p o n e n t to t h e o t h e r [4].
Glycopeptide preparation and purification. T h e o v o m u c o i d s u b m i t t e d to p r o n a s i c d i g e s t i o n w a s p r e v i o u s l y c a r b o x y m e t h y l a t e d i n o r d e r to i n c r e a s e t h e s p e e d of h y d r o l y s i s w h i c h a p p e a r s , i n this case, to o c c u r l0 t i m e s f a s t e r [20]. T h e c h e m i c a l m o d i f i c a t i o n of t h e g l y c o p r o t e i n does n o t i m p a i r its P~-like activity.
A 215 nn 489nm
~z~ m
mho
20
A
lo
50
100
150
200
fraclions
FIG_ 1. - - Chromatography of the mixture of glycopeptides on a P-cellulose column (26 × 2 cm) equilibrated in 0.02 N H~PO4 pH 2.45. Elution is performed by applying a gradient of ionic strength: 1° 150 ml. 0.02 N HaPO4, 2° 200/200 ml. 0.02 N HaPO4 containing O - 0.2 M KCI. The absorbancy of each fraction (3 ml.) is measured at 215 nm (e--o) and at 489 nm ( / k - - - A ) after a phenol-sulfuric reaction performed with 200 Ial aliquots. The increase of ionic strength is illustrated by the conductivity of the eluate ( 0 - - 0 ) . T h e u n r e t a r d e d p e a k o b t a i n e d o n t h e Biogel-P a c o l u m n c o n t a i n s all t h e g l y c o p e p t i d e s as s h o w n b y t h e p h e n o l - s u l f u r i c test. These f r a c t i o n s w e r e p o o l e d , l y o p h i l y z e d a n d s u b m i t t e d to 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 n a P-cellulose c o l u m n . T h e e l u t i o n p r o f i l e is s h o w n i n Fig. 1. O u t of six m a j o r p e a k s , o n l y o n e is free of a n y glycosidic
SPECIFIC BLOOD-GROUP Pt
583
r e s i d u e ; the others have been n a m e d P r l to Pr5 in the o r d e r of elution. The m o s t basic one, PrS, is also the m o s t active anti-P I inhibitor. T h a t is the r e a s o n w h y we have, at first, u n d e r t a k e n the study of the Pl-like antigenic s t r u c t u r e on this glycopeptide. The glycopeptide was f u r t h e r purified b y c h r o m a t o g r a p h y on DEAEcellulose (Fig. 2). I t a p p e a r s to be p u r e as checked by p a p e r electrophoresis at p H 2.4 in 1 M acetic acid.
FIG.
2. - - C h r o m a t o g r a p h y of Pr5 glycopeptide o n a DEAE-cellulose c o l u m n (15 × 1.5 cm) e q u i l i b r a t e d in a Tris-HC1 pH 8 buffer (0.015 M HC1 Tris). E l u t i o n is e n s u r e d by a g r a d i e n t of ionic s t r e n g t h f r o m 0 to 0.3 M NaCI in a m i x t u r e of 150/150 m l of b u f f e r . The a b s o r b a n c y of each f r a c t i o n (2 ml.) is m e a s u r e d at 215 n m (e--e). The p h e n o l - s u l f u r i c reaction a p p l i e d to 200 ~1. of a l i q u o t is r e a d at 489 n m ( ~ - - ' A ) . The c o n d u c t i v i t y of the eluate s h o w s the slope of the ionic s t r e n g t h increase ( Q - - G).
I
50
10D rraclions
Structure analysis of Pr5. AMINO ACID AND SUGAR COMPOSITION.
The osidic c o m p o s i t i o n of glycopeptide Pr5 d e t e r m i n e d by methanolysis is s u m m a r i z e d in Table I which also presents the a m i n o acid content of the peptidic moiety. TABLE I
A m i n o acid composition and carbohydrate content of glycopeptide Pr5 expressed as n u m b e r of residues per mole of glycopeptide.
AMIN0 ACID : His . . . . . . . . . . . . . . . . . . . . . . . . . ASX . . . . . . . . . . . . . . . . . . . . . . . . . . Pro . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1 1 0.5
(1) (1) (o-D
5.7 3 6.5
(6) (3) (7-8)
CARBOHYDRATE : Gal . . . . . . . . . . . . . . . . . . . . . . . . . . Man . . . . . . . . . . . . . . . . . . . . . . . . GIcNAc . . . . . . . . . . . . . . . . . . . . . .
584
FRANCOIS-GERARD Ch. and coll.
METHYLATION. T h e a n a l y s i s o f t h e m e t h y l a t e d d e r i v a t i v e s of Pr5 w a s c o n s i s t e n t w i t h g a l a c t o s e r e s i d u e s i n t e r m i n a l p o s i t i o n s i n c e a l a r g e a m o u n t of m e t h y l 2,3,4,6-tetra-O-methyl g a l a c t o s i d e c o u l d b e i d e n t i f i e d b y g.l.c. T h e p r e s e n c e o f b o t h m e t h y l 2,3,6,-tri-O-methyl g a l a c t o s i d e a n d m e t h y l 3,6-di-O-methyl-N-(acetyl) m e t h y l g l u c o s a m i n i d e d e n o t e s t h a t G a l a n d G l c N A c r e s i d u e s a r e s u b s t i t u t e d in t h e C 4 p o s i t i o n . T a b l e I I s u m m a r i z e s all t h e m e t h y l a t e d d e r i v a t i v e s t h a t c o u l d b e i d e n t i f i e d b y g.l.c. TABLE II Identification by g.l.c, and molar ratio of the O-methyl glycosides present in the Pr5 glycopeptide after methylation.
2, 2, 3, 2, 3 2 2, '3,
METHYLATEDMONOSACCHARIDES
MOLAR RATIO
3, 4, 6 - - tetra-O-methyl-galactoside . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 - - tri-O-methyl-galactoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 - - di-O-methyl rnannoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 - - di-O-methyl malanoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - mono-O-methyl mannoside . . . . . . . . . . . . . . . . . . . . . . . . . . - - InOlao-O-methyl mannoside . . . . . . . . . . . . . . . . . . . . . . . . . . 3, 4, 6 - - tetra-O-N-acetyl-rnethyl glucosaminide . . . . . . . . . . . . . . 6 - - di-O-acetyl-methyl glucosaminide . . . . . . . . . . . . . . . . . . . .
+ 3 1 1 1 1
+ 5
HYDRAZINOLYSIS. Hydrazinolysis followed by nitrous deamination specifically cleaves t h e G l c N A c 0 - g l y c o s i d i c l i n k a g e s , c o n v e r t i n g G l c N A c i n t o 2,5-anhydrom a n n o s e . W h e n t h i s l a t t e r p r o d u c t is r e d u c e d , it is i d e n t i f i e d as 2,5a n h y d r o m a n n i t o l in g.l.c. I n t h e p r o d u c t s l i b e r a t e d b y t h i s m e t h o d , a t r i s a c c h a r i d e w a s f i r s t s e p a r a t e d b y g.l.c, a n d t h e n i d e n t i f i e d b y m a s s s p e c t r o m e t r y as c o r r e s p o n d i n g to t h e s e q u e n c e : Gal-Gal-2,5anhydromannitol. ENZYMIC DIGESTION AND IOa-LIKE ACTIVITY. T h e ~ - g a l a c t o s i d a s e i s o l a t e d f r o m A s p e r g i l l u s N i g e r w a s p r o v e d to b e f r e e of a n y o t h e r g l y c o s i d a s i c a c t i v i t y b y t e s t i n g s e v e r a l s y n t h e t i c s u b s t r a t e s . I t s a c t i v i t y is e q u i v a l e n t to 0.2 ~ m o l e of p a r a - n i t r o - p h e n y l a - D - g a l a c t o p y r a n n o s i d e h y d r o l y z e d a t 37°C. p e r m i n u t e a n d p e r rag. of protein. A s i g n i f i c a n t loss o f P~-activity w a s o b s e r v e d a f t e r 120 h. of d i g e s t i o n u s i n g a 1/1 ( w / w ) e n z y m e t o p e p t i d e r a t i o . Fig. 3 s h o w s t h e e v o l u t i o n of t h e PI a c t i v i t y as a f u n c t i o n of t i m e . T h e p e r c e n t a g e of r e m a i n i n g a c t i v i t y , e x p r e s s e d as t h e c o n c e n t r a t i o n r e s p o n s i b l e f o r 50 % of h a e m a g g l u t i n a t i o n i n h i b i t i o n , d e c r e a s e s f o l l o w i n g a n e x p o n e n t i a l curve. T h e a n a l y s i s a f t e r m e t h a n o l y s i s of t h e p r o d u c t of d i g e s t i o n r e v e a l s a s i g n i f i c a n t l o s s of t h r e e g a l a c t o s e r e s i d u e s . F u r t h e r m o r e , m e t h y l a t i o n
SPECIFIC BLOOD-GROUP P1
585
of the e-galactosidase t r e a t e d glycopeptide showed a decrease of m e t h y l 2,3,6-tri-0-methyl galactoside whereas the a m o u n t of m e t h y l 2,3,4,6-tetra0-methyl galactoside r e m a i n s identical. This suggests the p r e s e n c e of Gal residues at the non reducing position, linked to o t h e r Gal residues by a ~-1,4 type linkages.
!' 0'5
FIG. 3, - - P1 activity oE g l y c o p e p t i d e Pr5 d i g e s t e d w i t h c~-ga|actosidase. T h e r e s u l t s a r e e x p r e s s e d as t h e fractional activity giving 50 % inhibition of haemagglu tination
0
(C 50 %).
I
l 20
40
GO
80
100
lime
(hou,s~
DISCUSSION
The N-glycosidically linked glycans of m o s t glycoproteins are built following a classical p e n t a s a c c h a r i d i c s t r u c t u r e involving a b r a n c h e d m a n n o s i d i c core, m a d e of three residues, linked to the peptidic chain by two sequential GlcNAc residues [21]. The two external m a n n o s e residues are p r e f e r e n t i a l sites for attaching two glycosidic chains. The possibility of m o r e b r a n c h e d glycannic s t r u c t u r e s cannot be, however, ruled out [22]. As turtle-dove o v o m u c o i d glycopeptide Pr5 is concerned, the presence of mono-, di- and tri-0-methyl m a n n o s i d e in the m e t h y l a t e d derivatives of Pr5, is in favour of a p o l y b r a n c h e d structure. Taking into account the P1 antigenic s t r u c t u r e s of b o t h h u m a n glycosphingolipid [7] and hydatid cyst glycoprotein [23], w e first looked for ~-Gal residues in t e r m i n a l position by using b o t h chemical and enzymatic m e t h o d s . The p r e s e n c e of tetra-0-methyl galactoside in the m e t h y l a t e d p r o d u c t s and the loss of P1 activity during ~-galactosidase digestion c o n f i r m s t i m u l t a n e o u s l y the existence of g-galactose at the non reducing end of the glycosidic chain. It m a y t h e r e f o r e be stated that ~-Gal is the i m m u n o d o m i n a n t sugar of the P~-like antigenic d e t e r m i n a n t of turtle-dove ovomucoid, as in the case of h u m a n erythrocyte and h y d a t i d antigen. Moreover, the lost of three Gal residues after exhaustive e-galactosidase t r e a t m e n t and the p r e s e n c e of a single p e a k c o r r e s p o n d i n g to the trisaccharide Gal-Gal-2,5 a n h y d r o m a n n i t o l , identified by m a s s s p e c t r o m e t r y in the hydrazinolysate of native Pr5 suggest t h a t three Pl-active trisaccharides are p r o b a b l y p r e s e n t per m o l e of glycopeptide.
586
FRANCOIS-GERARD
Ch. and coll.
Pooling all results o b t a i n e d b y chemical and enzymatical ways, we p r o p o s e the following s t r u c t u r e (Fig. 4) for the Pl-like antigenic d e t e r m i n a n t associated to the Pr5 glycopeptide of turtle-dove ovomucoid. I t w o u l d b e h a z a r d o u s to establish a definite s t r u c t u r e for the glycopeptide Pr5 as long as the o t h e r glycopeptidic s t r u c t u r e s of turtle-dove o v o m u c o i d are not yet c o m p l e t e l y elucidated. According to the analyses u n d e r t a k e n to c h a r a c t e r i z e the o t h e r glycopeptides, we t h i n k t h a t t h r e e different glycosylation sites t a k e place on the polypeptide chain of turtle-dove ovomucoid, each of t h e m p r o d u c i n g a Pl-active glycopeptide.
GIENAc
(1-4) GIcNAc (1--2)
Man
GIcNAc (1~4) Man (I~4)GIcNAc (x--4)G[cNAc ~ A s n
/-6,a) ¢=Ga[ (1-4) Gal (1-4) GIcNAc (1~2) Man GIcNAc /~4) GBI /--4) ~-Gal
~/(~-o)
(1-3)
G[cNAc I (1--4) Ga[
I,1-4)
-Gal
FIG. 4. - - H y p o t h e t i c a l s t r u c t u r e p r o p o s e d f o r o n e of t h e P1active g l y c o p e p t i d e s i s o l a t e d f r o m t u r t l e - d o v e o v o m u e o i d b y p r o n a s i c digestion.
RESUME L ' o v o m u c o i d e pr6sent dans le blanc d'.ceuf de tourterelle est une nouvelle s u b s t a n c e soluble d o n n a n t lieu ~ une rdaction crois4e avec l'antighne P~ humain. L ' o v o m u c o i d e est une glycoprot6ine de P.M. 29 400, c o n t e n a n t envir o n 20 % de rdsidus osidiques r6partis e n : m a n n o s e , galactose, NacEtyl glucosamine et acide N-acdtyl-neuraminique. C o m m e la glycoprot6ine du liquide de kyste hydatique, l'ovomucoide de tourterelle est un i m m u n o g h n e efficace p u i s q u e d'excellents s6rums anti-P 1 ont 6t4 obtenus p a r i m m u n i s a t i o n de lapins. P o u r c o n n a i t r e la s4quence osidique s p d c i f i q u e m e n t r e s p o n s a b l e de l'activit6 antig6nique P1, l ' o v o m u c o i d e de tourterelle a 6t6 fragmental par digestion pronasique. Les glycopeptides Pl-actifs ont 6t6 sdpar6s par filtration mol4culaire puis purifies p a r c h r o m a t o g r a p h i e sur 6changeurs d'ions. Le plus actif d ' e n t r e eux, le f r a g m e n t Pr 5, a 6t6 l'objet d'une r e c h e r c h e plus approfondie.
SPECIFIC BLOOD-GROUP PI
587
Les m 6 t h o d e s p h y s i c o - c h i m i q u e s telles la c h r o m a t o g r a p h i e e n p h a s e gazeuse et la s p e c t r o m 6 t r i e de m a s s e p e r m e t t e n t de c o n c l u r e q u ' u n e s 6 q u e n c e t r i s a c c h a r i d i q u e ~-Gal 1 ~ 4 ~-Gal 1 ~ 4 ~ GlcNAc i d e n t i q u e /~ celle q u i c a r a c t 6 r i s e l ' a n t i g 6 n e P~ 6 r y t h r o c y t a i r e , est e f f e c t i v e m e n t pr6s e n t e d a n s l ' o v o m u c o i d e de t o u r t e r e l l e . De p l u s , la d i g e s t i o n e n z y m a t i q u e p a r l ' ¢ - g a l a c t o s i d a s e , q u i e n t r a i n e u n e p e r t e de l ' a c t i v i t 6 P~-like, conf i r m e q u ' u n r 6 s i d u d'~-Gal est 6 g a l e m e n t le s u c r e i m m u n o d o m i n a n t d u d d t e r m i n a n t P1 de l ' o v o m u c o i d e . Les r e c h e r c h e s e n t r e p r i s e s p o u r c a r a c t 6 r i s e r les a u t r e s f r a g m e n t s Pl-actifs de l ' o v o m u c o i d e , a m 6 n e n t ~ p e n s e r q u ' i l existe 3 sites de glycos y l a t i o n p a r m o l 6 c u l e d ' o v o m u c o i d e , c h a c u n d ' e u x p r o d u i s a n t u n glycop e p t i d e P~-actif.
Request reprints f r o m : Ch. FRAN§OIS-GF;RARD, Universit~ de Li6ge, Laboratoire des Groupes Sanguins et de Transfusion, 41, rue Dos-Fanchon, B-4020 LI~?GE (Belgique).
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588
FRANCOIS-GERARD
Ch. and coll.
[11] DUBOlS M., GILLES K.A., HAMILTON J.K., REBERS P.A_ and SMITH F. - Colorimetric m e t h o d for d e t e r m i n a t i o n of sugars and released substances. Anal. Chem., 28, 350 (1956). [12] ZANETTA J.P., BRECKENRIDGE W . C . a n d VINCENTON G. - - Analysis of monosaccharides by g.l.c, of the O-methylglycosides as trifluoroacetate derivatives. Application to glycoproteins a n d glycolipids. J. Chromatogr., 69, 291 (1872). [13] BENSON J.V. a n d PATTERSONJ'.A. m Accelerated automatic chromatographic analysis of a m i n o acids on a spherical resin. Anal. Chem., 37, 1108 (1965). [14] HAKOMORIS.I. - - A rapid p e r m e t h y l a t i o n of glycolipid a n d polysaccharide catalyzed b y methyl sulfinyl c a r b a n i o n in dimetyl sulfoxide_ Y. Biochem., 55, 205 (1964). [15] FOURNET B. - - (Personal communication). [16] BAYARD B. a n d MONTREUIL J. - - Specific cleavage of N-acetyl glucosam i n i d i c linkages. Colloques Internationaux du C.N.R.S., 221, 209 (1973). [17] STRECKER G., FOURNET B., MONTREUIL ]'., DORLAND L., HAVERKAMP J. a n d VLIEGENTHART J.F.G. - - S t r u c t u r e of the three m a j o r fucosyl-glycoaspargines a c c u m u l a t i n g in the u r i n e of a p a t i e n t with fucosidosis. Biochimie, 60, 725 (1978). [18] BOUOUELET S., SPIK G. and MONTREUIL ft. - - Properties of a a-mannosidase from Aspergillus Niger. Biochim. Biophys. Acta, 522, 521 (1978). [19] ASTHON W.D. - - The logit t r a n s f o r m a t i o n with special reference to its uses in bioassay. Griffin's Statistical Monographs a n d Courses (Ed. Alan Stuart, 1972). [20] FRAN§OIs-GI~RARDCh. - - (Unpublished results). [21] MONTREUILJ. - - Recent data on the s t r u c t u r e of the c a r b o h y d r a t e moiety of glycoproteins. Metabolism and biological implications. Pure and applied Chemistry, 42, 431 (1975). [22] STRECKER G. and MONTREUIL J. - - Glycoproteins and glycoproteinoses. Biochimie, 61, 1199 (1979). [23] WATKINS W.M. a n d MORGAN W.T.J. - - Blood group P1 s u b s t a n c e : II I m m u n o l o g i c a l properties. Proc. 9th Congr. int. Soc. Blood Transf. Mexico, 1962. S. Karger B~tle 230 (1964).