The Tk antigenic determinant studies of Tk-activated red blood cells with endoglycosidases

Blood Transfusion and Immunohaematology Tome XXIV. - - N° 1. - - 1981

109

The antigenic determinant studies o f Tk.activated red blood cells with endoglycosidases by C. D O I N E L , J.M. R U F I N and G. A N D R E U

INTRODUCTION

T

k polyagglutination is a red cell m e m b r a n e modification associated with a bacterial infection [2, 3]. The red blood cells f r o m a case of Bacteroides fragilis septicemia were f o u n d to be Tk polyagglutinable [3] and the Tk t r a n s f o r m a t i o n can be induced in vitro by culture s u p e r n a t a n t s f r o m some strains of Bacteroides /ragitis [13, 14]. Tk ceils are agglutinated by m o s t AB sera, Arachis hypogae and Bandeiraea simplici/olia (BS II) lectins. These reactivities are enhanced after t r e a t m e n t with papain or ficin [5, 8].

In vivo Tk activation has been f o u n d alone [2, 3, 15] or associated with T, acquired B, Va polyagglutinabilities [5, 13, 17]. Tk and acquired B polyagglutinability antibodies are different as shown by cross absorption and elution tests p e r f o r m e d with cells, bearing specific antigenic d e t e r m i n a n t s [1]. Modifications of ABH antigens [1, 15] and also the destruction of I and i antigens o n T k activated red cells [1, 8] have been described. The alternative hypothesis for the change of ABH, Ii antigenicity and the u n m a s k i n g of Tk activity following t r e a t m e n t of h u m a n red blood cells with B. /ragilis culture s u p e r n a t a n t s is the action of several exoglycosidase activities o r one endoglycosidase. Arguments for the latter hypothesis were s u p p o r t e d by the red cell modification p r o d u c e d in vitro by an endo-~-galactosidase isolated f r o m Escherichia freundii

This work was supported by a grant from DGRST (MB -- P 256).

110

DO1NEL C. and coll.

a n d k i n d l y d o n a t e d b y D r M.N. FUKUDA ( B i o c h e m i c a l O n c o l o g y - F r e d H u t c h i n s o n Cancer R e s e a r c h Center, Seattle, Wash.). Moreover, the purification and c h a r a c t e r i z a t i o n of the glycosidases f r o m culture supern a t a n t of B . f r a g i l i s w e r e s t a r t e d .

T k polyagglutination i n d u c e d in vitro by an endo-~-galactosidase Purified endo-~-galactosidase from Escherichia freundii modified s p e c i f i c r e d cell m e m b r a n e g l y c o c o n j u g a t e s , a n d p r o d u c e d a d e c r e a s e o f t h e A B H r e a c t i v i t i e s a n d a n i m p o r t a n t r e d u c t i o n o f I a n d i antig e n i c i t i e s . T h e s e a n t i g e n i c c h a n g e s of h u m a n e r y t h r o c y t e s a n t i g e n i c i t y w e r e s i m i l a r to t h e s e o b s e r v e d a f t e r t r e a t m e n t b y B . f r a g i l i s s u p e r n a t a n t s ( T a b l e I). TABLE I Serological modifications of I positive cells following treatment with B. fragilis culture supernatant or endo-~-galaetosidase of E. freundii. UNTREATEDRBC

B. fragilis SUPERNATANT

66 * 86 30 **

51 * 20 0

Anti-B . . . . . . . . . . . . . Anti-I . . . . . . . . . . . . . Anti-i . . . . . . . . . . . . . AB serum (anti-Tk) BS II lectin . . . . . .

E. [reundii ENDO-~-GALACTOSIDASE 63 * 51 0

31 118

2 7

45 118

*: Agglutination score. Test on the papainized red cells. **: i reactivity of I positive cells is unmasked by papain treatment [7].

T h e d i f f e r e n t i a l c h a n g e of e r y t h r o c y t e I a n d i r e a c t i v i t i e s ( T a b l e I I ) suggested that the linear carbohydrate chains were hydrolyzed more readily than the branched structures.

TABLE II I and i reactivities of red cells treated with B. fragilis culture supernatants. OI

Oi adult

I activities *

i activities *

100 %

100 %

39 %

0%

RED CELLS

non treated (saline) treated (v/v, 2h, 37°C)

*: I and i blood group activities evaluated with anti-I (ABG) and anti-i (Fou.) by quantitative haemagglutination method as previously described [6].

Tk ANTIGENIC DETERMINANT STUDIES

111

FUKUDA et al. [12] h a v e also s h o w n a n i n c r e a s e of t h e r e a c t i v i t y of r e d cells t r e a t e d w i t h E s c h . f r e u n d i i w i t h t h e a n t i - l a c t o - N - t r i o s y l c e r a m i d e . This e n h a n c e d a g g l u t i n a b i l i t y w a s o b s e r v e d w i t h n o r m a l I a d u l t , Oi a d u l t a n d c o r d Oi e r y t h r o c y t e s o n l y w h e n t h e y w e r e t r e a t e d w i t h t r y p s i n e a n d endoq3-galactosidase. T h e r e s u l t is c o m p a t i b l e w i t h t h e u n m a s k i n g of t h e GlcNAc-~3-(1-3)-Gal-~-(1-4) s t r u c t u r e as t h e effect of b o t h e n z y m a t i c t r e a t m e n t . T h e Tk r e a c t i v i t y , i.e. t h e a g g l u t i n a b i l i t y b y t h e B S I I l e c t i n [17, 4] is p r o b a b l y d u e to t h e a c c e s s i b i l i t y of this s t r u c t u r e o n t h e e n d o @ g a l a c t o s i d a s e t r e a t e d cells. I n d e e d t h e BS I I a g g l u t i n a t i o n w a s i n h i b i t e d b y N - a c e t y l - g l u c o s a m i n e [16, 17, 20] a n d b y oligosaccharides with a terminal N-acetyl-glucosamine residue (Table III). TABLE I I I

Inhibition of the Tk activated red blood cells and BS II lectin haemagglutination reaction by various sugars and glycoconjugates.

INHIBITORS

CONCENTRATION FOR TOTAL INHIBITION

~g/100 D1 Glucose . . . . . . . . . . . . . . . . . . . . . . . . . Galactose . . . . . . . . . . . . . . . . . . . . . . . Fucose . . . . . . . . . . . . . . . . . . . . . . . . . . NAc-galactosamine . . . . . . . . . . . . . . NAc-glucosamine . . . . . . . . . . . . . . .

> > > >

1000 1000 1000 1000 1000

PNP-~-Gal . . . . . . . . . . . . . . . . . . . . . . PNP-~-Gal . . . . . . . . . . . . . . . . . . . . . . PNP-a-NAcGal . . . . . . . . . . . . . . . . . . PNP-v:NAcGlc . . . . . . . . . . . . . . . . . . PNP-~-NAcGlc . . . . . . . . . . . . . . . . . .

> 1000 > 1000 850 1,7 102

GlcNAc-~-(1 ~ 3) Gal . . . . . . . . . . . . GIcNAc-~-(1 ~ 6)

80

Gal-~-(1 ~ 3)-GIcNAc-~-(1~ 3) Ga] > 800 GlcNAc-~-(1 ~ 6)

Purification and characterization of glycosidases from B-fragilis INGLIS et al. [15] f o u n d s e v e r a l g l y c o s i d a s e s i n t h e c u l t u r e m e d i u m of B. fragilis. I n t h e s u p e r n a t a n t s of B. fragilis s t r a i n s k i n d l y p r o v i d e d b y A.A.B. MITCHELL a n d G. INGLIS w e f o u n d a n ~-D-Galactosidase a n d a ~ - D - N A c - G l u c o s a m i n i d a s e b u t n o n e u r a m i n i d a s e n o r p r o t e o l y t i c activities ( T a b l e IV). T h e p r o c e d u r e of p u r i f i c a t i o n is s h o w n i n f i g u r e 1. All t h e steps w e r e p e r f o r m e d a t 4°C. T h e p r e c i p i t a t e I I I w a s free of ~-Galactosidase. I n t h e D E A E - S e p h a d e x c h r o m a t o g r a p h y step, t h e e n z y m a t i c f r a c t i o n s w h i c h i n d u c e d t h e Tk-activation w e r e u n a d s o r b e d w h i l e t h e exoglycosidase a c t i v i t i e s w e r e e l u t e d w i t h a c o n t i n u o u s salt g r a d i e n t o b t a i n e d w i t h 0.5M NaC1.

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DOINEL C. and coll.

TABLEIV Exo-glycosidase activities in B. fragilis culture fluid. p-nitrophenyl-glycosides

1 U = 1 izmole/ml/min

~z-D-Mannose . . . . . . . . . . . . . . . . . . . . 3-D-Glucose . . . . . . . . . . . . . . . . . . . . . ]-D-NAc Glucosamine . . . . . . . . . . ]-D-Galactose . . . . . . . . . . . . . . . . . . . .-D-Galactose . . . . . . . . . . . . . . . . . . . ~-D-Nac Galactosamine . . . . . . . . 6~-L-Fucose . . . . . . . . . . . . . . . . . . . . . .

0 0 0.2 0 0.8 0 0

Neuraminyllactose . . . . . . . . . . . . . .

0*

* : No free sialic acid detected by the thiobarbituric acid method [19]. Culture Supernatants Bacteroides fragilis

i

Fractional precipitation by (NH4)2 SO4: 4C, 18~

1.5 M I

,

1

10.000 g

Sl

I

1

II

2. 5 M S II 3.8M

ill

7 1

S Ill

DEAE~Sephadex A-25 Chromatoqraphy : 0.01 M Tris ; pH 7.5

1 1

Gel-filtration Sephadex G-100:0.1 M Tris, 0.2M NaCI;pH 7.}

Concentration, Dialysis, Storage --196"C

FIG. 1. - - Purification procedure of endo-glycosidase from B. fragilis. T h r o u g h this p r o c e d u r e t h e e n z y m e w a s p u r i f i e d 55-fold w i t h 71 r e c o v e r i e s ( T a b l e V). U s i n g p - n i t r o - p h e n y l g l y c o s i d e s t h e e n z y m e prep a r a t i o n w a s free f r o m t h e f o l l o w i n g g l y c o s i d a s e s : ~-D-galactosidase, B-D-Galactosidase, B-D-Glucosidase, B-D-NAc-Glucosaminidase, ~-D-NAcGalactosaminidase, a-L-Fucosidase and a-D-manosidase. Pooled actived f r a c t i o n s w e r e d i a l y z e d a g a i n s t 0.15M NaC1, p H 6.0 a n d s t o r e d i n l i q u i d nitrogen. T h e c h a r a c t e r i z a t i o n of t h e e n z y m e p r e p a r a t i o n w i t h h u m a n r e d b l o o d cells, h u m a n b l o o d g r o u p g l y c o p r o t e i n , o l i g o s a c c h a r i d e s a n d b o v i n e c o r n e a l k e r a t a n s u l f a t e is i n p r o g r e s s . T h e e n z y m e h y d r o l y s e s

Tk ANTIGENIC DETERMINANT STUDIES

113

TABLE V Summary of purification.

STEPS

TOTAL ACTIVITY SCORE

Culture fluid . . . . . . . . Ammonium sulfate ~, DEAE Sephadex ... Sephadex G 100 . . . .

30800 27700 22400 22050

TOTAL PROTEINS mg 40,7 5,9

1,05 0,54

SPECIFIC * ACTIVITY SCORE/mg

RECOVERY

756 4625 21330 40835

100 90 73 71

%

*: Expressed as agglutination score on Tk activated red cells.

0 hour

10 18hours

1~

20

25

3JO 3H I(Ma) trisaccharide J4C glucose

o

7,. o

/ / 1'0

I]5 2'0 2'5 3'0 fractions number

3~5

FIG. 2. - - Chromatogramm of Sephadex G 50 gel filtration of bovine corneal keratan sulfate and its endo-glycosidase digestion products. Keratan sulfate (0,1 rag) in 0,2M Acetate buffer pH 6.0 was incubated with purified enzyme fraction (200 I~l, 25 Ixg) at 37°C for 18 h. Incubation mixture with and without enzyme wag applied to a colunm (1,5 x 30 cm) and eluted with 0,15 NaC1. Fractions of 1,5 ml were isolated and assayed for hexoses (10).

the H active blood group ovarian substance. As s h o w n i n f i g u r e 2 t h e digestion products of keratan sulfate can be separated into several f r a c t i o n s b y g e l f i l t r a t i o n o n S e p h a d e x G 50 s u p e r f i n e e q u i l i b r a t e d w i t h 0,2M NaC1. T h e s m a l l e s t d i g e s t i o n p r o d u c t is p r o b a b l y a d i s a c c h a r i d e w h i c h is e l u t e d b e t w e e n t h e Gal-~-(1-4)-GlcNAc-~-(1-6)-Gal t r i s a c c h a r i d e a n d t h e G l u c o s e ( F i g . 2). Other investigations are in progress to

DO1NEL C. and coll.

114

characterize these different oligossacharidic fractions and to identify the red cell glycoproteins which were modified by the enzyme treatment. CONCLUSION After treatment with the endo-~-gaIactosidase from Esch. lreundii or with an enzymatic fraction extracted from the culture fluid of B. ~ragilis and free of exoglycosidases, the human red blood cells are Tk activated. This specific agglutination with the BS II lectin is inhibited by N-acetyl-glucosamine and oligo-saccharides with terminal N-acetylglucosamine residue. These results and others previously described by ANDREU et al. [1] exclude the identity between Tk and acquired B polyagglutinations. As suggested by : a) The parallelism between B. [ragilis and Esch. [reundii endoglycosidases concerning the antigenic changes of human red blood cells and the gel chromatography profile of the digestion products of keratan sulfate [10], b) the enzyme specificity of the endo-~-galactosidase of Esch. Ireundii which hydrolyses the oligosaccharides having the common structure R-GlcNAc-B-(1-3)-Gal-~-(1-4)-Glc (or G1NAc) and creates a terminal N-acetylglucosamine-~-(1-3) structure [11], c) the structure of the i- and I-active glycolipids, Gal-~-(1-4)-GlcNAc~-(1-3)-Gal-~-(1-4)-GlcNAc-~-(1-3)-Gal and GaI-~-(1-4)-GlcNAc-~-(1-3) [Gal-~(1-4)-GlcNAc-~-(1-6)] Gal respectively [18, 9], the main antigenic determinant of Tk reactivity is a terminal N-acetylglucosamine residue; RESUME Les globules rouges humains trait6s in vitro par l'endo-~-galactosidase d'Esch, freundii [8] ou avec une fraction enzymatique purifi6e h partir des surnageants de culture de B. fragilis et exempte d'activit6s exoglycosidases deviennent Tk activ6es. Ces h6maties transform6es sont agglutinables par la lectine BS II de Bandeiraea simplicifolia. Cette agglutination est inhib6e par la N-ac6tyl-glucosamine et les olig0saccharides ayant un groupement ~-N-ac6tyl-glucosamine terminal. Aprbs purification l'enzyme de B. Iragilis hydrolyse les keratan sulfates et donne un profil chomatographique proche de ceux observ6s avec l'endo-~-galactosidase d'Esch, freundii. Les produits form6s sont en cours d'identification. Parall~lement les deux enzymes d6truisent les antig~nes I et i 6rythrocytaires. Si l'on consid6re, d'nne part les structures des glycolipides 6rythrocytaires i et I, Gal%(1-4)-GlcNAc-~-(1-3)-Gal-~-(1-4)-GlcNAc%(1-3)-Gal et Gal-~-(1-4)GlcNAc-~-(1-3) [Gal-~-(1-4)-GlcNAc- ~-(1-6)]-Gal respectivement [18, 9], d'autre part la sp6cificit6 de l'endo-~-galactosidase d'Esch.

Tk ANTIGENIC DETERMINANT STUDIES

115

[reundii q u i h y d r o l y s e les o l i g o s a c c h a r i d e s a y a n t u n e s t r u c t u r e R-GlcNAc~-(1-3)-Ga143-(1-4)-Glc ( o u GlcNAc) et cr6e u n e s t r u c t u r e avec u n g r o u p e m e n t t e r m i n a l N - a c d t y l g l u c o s a m i n e 9-(1-3), il est p r o b a b l e q u e le d6term i n a n t a n t i g 6 n i q u e m a j e u r d 6 f i n i s s a n t la p o l y a g g l u t i n a b i l i t 6 Tk est UH groupement N-ac6tyl-glucosamine terminal. Request reprints f r o m : Dr. C. DOINEL C.N.T.S. Groupe INSERM U 76 75571 PARIS Cedex 12. ACKNOWLEDGEMENTS, We wish to t h a n k Dr H. DEBRAY for providing us with the BS I I lectin, Dr G. INGLIS for the gift of B. [ragilis culture s u p e r n a t a n t s as well as Dr M.N. FUKUDA for the gift of Esch. freundii endo-~-galactosidase a n d k e r a t a n sulfate. -

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REFERENCES [1] ANDREU G., DOINEL C., CARTRON J.P., MATIVET S . a n d SALMONC. - - I n d u c t i o n of Tk polyagglutination by Bacteroides fragilis culture supernatants. Associated modifications of ABH a n d Ii antigens. Blood T r a n s f u s i o n and I m m u n o h a e m a t o l o g y , 22, 551, 1979. [2] BIRD G.W.G. a n d WINGHAMJ. - - Tk :" A new form of red cell polyagglutination. Brit. J. Haematology, 23, 759, 1972. [3] BIRD G.W.G., WlNGHAMJ., INGLIS G. and MITCHELL A.A.B. - - Tk polyagglut i n a t i o n in Bacteroides fragilis septicemia. Lancet, i, 286, 1975. [4] BIRD G.N.G., WINGHAMJ., BECK M.L., PIERCE S.R., OATES G.D. and POLLOCK A. - - Th : A new form of erythrocyte polyagglutination. Lancet, 1215, 1978. [4] BYRNE V., BROWN A., ROPARS C. a n d MOORE B.P.L. - - Acquired B antigen, Tk activation a n d A1 destroying enzyme activity in a patient with septicemia. Vox Sang., 36, 208, 1979. [6] DOINEL C., ROPARS C. a n d SALMON C. - - A correction to the autoanalyser data for q u a n t i t a t i v e agglutination m e a s u r e m e n t s . Vox Sang., 28, 376, 1975. [7] DOINEL C., ROPARS C. a n d SALMON C. - - Effects of proteolytic enzymes a n d n e u r a m i n i d a s e on the I and i erythrocyte antigen site. Quantitative and t h e r m o d y n a m i c studies. Immunology, 34, 653, 1978. [8] DOINEL C., ANDREU G., CARTRON J.P., SALMON C. and FUKUDA M.N. - - Tk polyagglutination produced in vitro by a n endo-~3-galactosidase. Vox Sang., 38, 94, 1980. [9] FEIZI T., CHILDS R.A., WATANABEK. and HAKOMORI S. - - Three types of blood group I specificity a m o n g monoclonal anti-I auto-antibodies revealed by analogues of a b r a n c h e d erythrocyte glycolipid. J. Exp. Med., 149, 975, 1979. [10] FUKUDAM.N. a n d MATSUMURAG. - - Endo-~-galactosidase of Escherichia Ireundii. Purification and endo-glycosidic action on k e r a t a n sulfates, oligosaccharides and blood group active glycoprotein. J. Biol. Chem., 251, 6218, 1976. [11] FUKUDAM.N., WATANABEK. a n d HAKOMORIS. - - Release of oligosaccharides from various glycosphingolipids by endo-~-galactosidase. J. Biol. Chem., 253, 6814, 1978.

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[12] FUKUDAM.N., I~UKUDAM. a n d HOKOMORIS. - - Cell surface modification by endo-~-galactosidase: Change of blood group activities a n d release of oligosaccharides from glycoproteins a n d glycosphingolipids of h u m a n erythrocytes. J. Biol. Chem., 254, 5458, 1979. [13] INGLIS G., BIRD G.W.G., MITCHELL A.A.B., MILNE G.R. a n d WINGHAM J. - Erythrocyte polyagglutination showing properties of b o t h T a n d Tk, p r o b a b l y induced b y Bacteroides fragilis infection. Vox Sang., 28, 314, 1975. [14] INGLIS G., BIRD G.W.G., MITCHELL A.A.B., MILNE G.R. a n d WINGHAM J. - Effect of Bacteroides fragilis on the h u m a n erythrocyte m e m b r a n e . Pathogenesis of Tk polyagglutination. J. Clin. Path., 28, 964, 1975. [15] INGLIS G., BIRD G.W.G., MITCHELL A.A.B. a n d WINGHAMft. - - Tk polyagglut i n a t i o n associated with reduced A a n d H activity. Vox Sang., 35, 370, 1978. [16] IYER P.N., WILKINSON K. a n d GOLDSTEIN I.J. - - An N-acetyl-D-glucosamine b i n d i n g lectin from Bandeiraea simplicifoIia seeds. Arch. Biochem. Biophys., •77, 330, 1976. [17] JUDD W.J., BECK M.L., HICKLIN B.L., IYER P.N.S. a n d GOLOSTEIN I.J. - BS II l e c t i n : A second h e m a g g l u t i n i n isolated from Bandeiraea simplicifolia seeds with affinity for type I I I polyagglutinable Ted cells. Vox Sang., 33, 246, 1977. [18] NIEMANN H., WATANABE K., HAKOMORI S., CHILDS R.A. and FEIZI T. - Blood group i a n d I activities of lacto-N-norhexaosylceramide a n d its a n a l o g u e s : The s t r u c t u r a l r e q u i r e m e n t s for i specificities. Biochem. Biophys. Res. Commun., 81, 1286, 1978. [19] WARRENL. The t h i o b a r b i t u r i c acid assay of sialic acids. J. Biol. Chem., 234, 1971, 1959. [20] WOOD C., KABATE.A., EBISU S. a n d GOLDSTEIN I.ff. - - An i m m u n o c h e m i c a l study of the c o m b i n i n g sites of the second lectin isolated f r o m Bandeiraea simplicifolia (BS II). Ann. Immunol. Inst. Pasteur, 129, 143, 1978. -

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