Molecular nature of the blood-group ABH antigens of the human erythrocyte membrane

Blood Transfusion and Immunohaematology Tome XXIII. - - N ° 5. - - 1980

545

Molecular nature of the blood-group ABH antigens of the human erythrocyte membrane by Jukka F I N N E , T o m K R U S I U S , H e i k k i RAUVALA and Johan JARNEFELT D e p a r t m e n t of Medical Chemistry, University of HELSlNKI (Finland).

T HEo l ibgl oo soadc-cghr oa ruipd eA Bs Ht r uscpt eucr ei fsi c ioft i e sg layr ec odceotnejrumg iant ee sd

by certain terminal p r e s e n t in v a r i o u s b o d y f l u i d s a n d t i s s u e s [1]. W h i l e m o s t of t h e w o r k c o n c e r n i n g t h e chemistry of the determinants was carried out on soluble glycoproteins, the m o l e c u l a r n a t u r e of t h e a n t i g e n s p r e s e n t o n t h e e r y t h r o c y t e m e m b r a n e h a s b e e n a m a t t e r of d e b a t e . T h e f i r s t c o m p o n e n t s of t h e e r y t h r o c y t e m e m b r a n e s h o w n b y chem i c a l m e t h o d s to h a v e b l o o d - g r o u p d e t e r m i n a n t s w e r e g l y c o l i p i d s of r e l a t i v e l y s m a l l size ( r e v i e w e d in [2]). L a t e r a n e w c l a s s of g l y c o l i p i d s was d e s c r i b e d , t h e p o l y g l y c o s y l c e r a m i d e s , w h i c h c o n t a i n a c a r b o h y d r a t e m o i e t y o f v e r y l a r g e size [3]. T h e h i g h a m o u n t of c a r b o h y d r a t e gives t h e s e g l y c o l i p i d s u n e x p e c t e d p r o p e r t i e s , s u c h as h i g h w a t e r - s o l u b i l i t y [4]. A l t h o u g h s o m e e v i d e n c e h a d b e e n p r e s e n t e d f o r the o c c u r r e n c e of b l o o d - g r o u p A B H d e t e r m i n a n t s a l s o in t h e g l y c o p r o t e i n s of t h e e r y t h r o c y t e m e m b r a n e [5-7], it w a s o f t e n s u g g e s t e d t h a t s u c h o b s e r v a t i o n s c o u l d b e d u e to c o n t a m i n a t i o n b y t h e w a t e r - s o l u b l e g l y c o l i p i d s , a n d t h a t t h e p r e d o m i n a n t , if n o t e x c l u s i v e , c a r r i e r s of t h e ABH determinants were glycolipids. I n t h e w o r k s u m m a r i z e d b e l o w w e w e r e a b l e to d e m o n s t r a t e t h a t n o t o n l y g l y c o l i p i d s b u t a l s o g l y c o p r o t e i n s of t h e e r y t h r o c y t e m e m b r a n e contain unexpectedly large carbohydrate moieties, called polyglycosyl chains. T h e s e c h a i n s a l s o c o n t a i n A B H d e t e r m i n a n t s a n d in a n a m o u n t even h i g h e r t h a n t h e g l y c o l i p i d s . F u r t h e r m o r e t h e d e t e r m i n a n t s w e r e s h o w n to o c c u r in s e v e r a l d i f f e r e n t g l y c o p r o t e i n s .

71oa puv -Vld ~I~tDI-IO~t

546

ISOLATION OF POLYGLYGOSYL P E P T I D E S W I T H BLOOD-GROUP ACTIVITY

F o r the s t u d y of the possible o c c u r r e n c e of blood-group ABH d e t e r m i n a n t s in the glycoproteins of the e r y t h r o c y t e m e m b r a n e we have u s e d the a p p r o a c h of p r e p a r i n g glycopeptides b y extensive proteolytic digestion. With this m e t h o d the solubilization of the proteinb o u n d c a r b o h y d r a t e is essentially quantitative, which is not the case, w h e n s o m e c o m m o n l y u s e d m e t h o d s designed to extract the glycoproteins f r o m the e r y t h r o c y t e m e m b r a n e [5, 6], are used. I m p o r t a n t c o m p o n e n t s , like the b a n d 3 and b a n d 4.5 proteins r e m a i n insoluble. F u r t h e r m o r e , the p r e p a r a t i o n of glycopeptides m a k e s possible the isolation and f r a c t i o n a t i o n of the individual c a r b o h y d r a t e moieties [8]. I n lectin affinity c h r o m a t o g r a p h y , using the anti-A, anti-B lectin of Bandeiraea simplicifoIia, it was f o u n d t h a t a fraction of the glycopeptides p r e p a r e d f r o m h u m a n blood-group A or B e r y t h r o c y t e s i n t e r a c t e d specifically w i t h the lectin [9] (Fig. 1). Analysis of the glycopeptides b y gel filtration indicated very large m o l e c u l a r size (Fig. 2).

5 mM 500mM

200 Z

[3_ (D

100

FRACTION

NUMBER

FIG. 1. -- Affinity chromatography of total glycopeptides from blood-group B erythrocytes on a Sepharose column containing the Bandeiraea simplici[olia lectin I. The bloodgroup active fraction is displaced by 5 mM galactose. Before chromatography, the glycopeptides had been N-(aH) acetylated in their peptide moiety. From ref. [9]. In h e m a g g l u t i n a t i o n inhibition assays, the glycopeptides exhibited the expected blood-group A and B activities. Chemical analyses revealed t h a t the molecules f r o m blood-group A b u t not B c o n t a i n e d N-acetylg a l a c t o s a m i n e ( d e t e r m i n a n t of A) w h e r e a s the a m o u n t of galactose ( d e t e r m i n a n t of B) o c c u r r e d c o r r e s p o n d i n g l y in higher a m o u n t in glycopeptides f r o m B e r y t h r o c y t e s [9]. Detailed chemical studies including specific d e g r a d a t i o n m e t h o d s [10, 11] indicated the general p r o p e r t i e s of the substances, as s u m m a r i z e d in Fig. 3. The c a r b o h y d r a t e - p e p t i d e linkage s e e m e d to be of the Nglycosidic type, as indicated b y its stability to mild alkali t r e a t m e n t , and b y the p r o d u c t i o n of glucosaminitol u n d e r strong alkaline condi-

547

BLOOD GROUP A B H A N T I G E N S

tions in the presence of borohydride. Aspartic acid was the main amino acid component after hydrolysis. That the compounds were not glycolipids was indicated by the absence of sphingosine, fatty acids and glucose, which are integral components of glycosphingolipids. The major part of the molecules consists of the repeating disaccharide galactosyl-N-acetylglucosamine. They thus resemble keratan sulphate, the main (and perhaps only) difference being the absence of sulphate. The terminal sequences contain structures commonly found in g l y c o p r o t e i n s o f g l y c o l i p i d s . B e c a u s e o f t h e s i m i l a r i t y of t h e s e

BD DE

FE TR ATR

200 2 el_ (D

100

i

i

20 30 4'0 FRACTION N U M B E R

FIG. 2. - - Gel filtration of blood-group active polyglycosyl peptides from A and B erythrocytes on a column of Sephadex G-50. The compounds were eluted between the positions of Dextran Mr 10000 (DE) and the void volume (BD) of the c o l u m n ; no material was found at the positions of the ~, conventional ,, glycopeptides from fetuin (FE), transferrin (TR) and asialotransferrin (ATR). From ref. [9].

Peripheral branches (4-11 nml/mo]) I I1

('ore structure

GIcNAc ( i l l Gal /~I--4)G cNAc(~ --

III

AcNeu(a2--3)Gal{~l --4)GlcNAc(fil -

IV

AcNeu(c~2-6JGal(~l

V

Repeating saccharide structure (5 - 1 4 m o I / m o l )

4)GlcNAc(fll -

Gal(~1-4)GlcNAc(~I 2

L - 3 ) G a l ( ~ 1-4)GicNAc(j~ 1 -

r

•

rMan/

I

~ GlcNAc--Asn

I

FucM \'I

GaINAc(a] - - 3 ) G a l ( f l 1 - 4 ) G I c N A c ( ~ I -" 2

I

Fuca 1 \II

Gal(a l - 3 ) G a [ ( / } 1 - 4 ) G I c N A c ( ~ I o

I Fuca I

FIG. 3. - - Proposed general structure of the polyglycosyl peptides. The structure of the core portion has not yet been established; whether or not there is a chitobiose unit at the proximal end is not known. From ref. [10].

548

F I N N E J. and coll.

glycOpelstides to t h e large g l y c o l i p i d s ( p o l y g l y c o s y l c e r a m i d e s ) , they w e r e called p o l y g l y c o s y l p e p t i d e s . lectin T h e f r a c t i o n of g l y c o p e p t i d e s n o t b o u n d to t h e B a n d e i r a e a w a s f o u n d to also c o n t a i n p o l y g l y c o s y l p e p t i d e s , a n d i n a n a m o u n t a p p r o x i m a t e l y f o u r t i m e s as high. T h e s e m o l e c u l e s d i d n o t c o n t a i n b l o o d - g r o u p d e t e r m i n a n t s . I n t e r e s t i n g l y t h e y w e r e of s l i g t h l y s m a l l e r size, c o n t a i n i n g a p p r o x i m a t e l y a n a v e r a g e of 35-50 s u g a r r e s i d u e s per m o l e c u l e , as c o m p a r e d to the a v e r a g e of 50-55 r e s i d u e s e s t i m a t e d for t h e b l o o d - g r o u p active s u b f r a c t i o n . T h e b i o c h e m i c a l e x p l a n a t i o n for this d i f f e r e n c e is n o t k n o w n at p r e s e n t .

I D E N T 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 S C A R R Y I N G T H E BLOOD-GROUP D E T E R M I N A N T S F o r t h e i d e n t 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 c o m p o n e n t s c a r r y i n g bloodg r o u p A B H d e t e r m i n a n t s , t h e t e c h n i q u e of b i n d i n g r a d i o a c t i v e l y l a b e l l e d l e c t i n to e l e c t r o p h o r e t i c a l l y s e p a r a t e d p r o t e i n i n p o l y a c r y l a m i d e gel w a s u s e d . T h e c o m p o n e n t s b i n d i n g t h e l e c t i n are v i s u a l i z e d b y autor a d i o g r a p h y . Fig. 4 s h o w s the r e s u l t of s u c h a n a n a l y s i s u s i n g the

7

A~ -A2 B

B ABAB 0

0

/'~i!!ii:

2-

34.1_ 4.2-

-PAS-1

[

Z,.5

-PAS-2

Dye front--

......

-PAS-3

FIG. 4. - - Blood-group A and B active components of the erythrocyte membrane. Each sample containing the same amount of membrane protein of the bloodgroups indicated were subjected to electrophoresis in polyacrylamide gels in the presence of SDS. Visualization of blood-group-active components was carried out by using ~2aI-labelled B a n d e i r a e a lectin and autoradiography. On the left is shown the gel pattern as stained for protein. From ref. [12].

549

BLOOD GROUP ABH ANTIGENS

~SI-labelled B a n d e i r a e a lectin [12]. Each sample contained the same amount of m e m b r a n e protein, the only difference being the blood-group. The blood-group dependence of the lectin binding d e m o n s t r a t e d that the components revealed contained blood-group determinants. Because of this internal control, unspecific adsorption, or binding to other sugar structures, can be excluded. The binding to A t samples was significantly higher than to B with the particular b a t c h of lectin used (the ratio of the B a n d e i r a e a isolectins is k n o w n to vary from seed to seed [13]). From the p a t t e r n of lectin-binding it can be concluded that in the h u m a n e r y t h r o c y t e m e m b r a n e all m a j o r glycoprotein c o m p o n e n t s (band 3, b a n d 4.5 and the sialoglycoproteins) as well as possibly several minor unidentified components, are carriers of blood-group ABH determinants. A similar analysis was also carried out using 12SI-labelled anti-H lectin from L o t u s t e t r a g o n o I o b u s [12]. As expected, this lectin b o u n d only to c o m p o n e n t s of blood-group O and A2 erythrocytes. As a difference to the B a n d e i r a e a lectin the sialoglycoproteins in the PAS-bands did not bind this lectin. This is most likely explained by the fact that the L o t u s lectin binds only to ~,, type 2 ,, H-determinants [14]. The sialoglycoproteins are k n o w n to contain determinants of another type in the alkali-labile chains [15]. The c o m p o n e n t s binding L o t u s lectin (band 3, b a n d 4.5 and polyglycosyl ceramides) are therefore indicated to have determinants of , which is in accordance with the chemical data on polyglycosyl peptides [10, 11] and polyglycosyl ceramides [3], respectively. That bands 3 and 4.5 contain polyglycosyl chains, is indicated by their susceptibility to endo-~-galactosidase digestion [16, 17]. It is thus possible to differentiate between two groups of blood-group d e t e r m i n a n t containing glycoconjugates by using lectins with different binding specificities. DISTRIBUTION IN DIFFERENT

OF A B H D E T E R M I N A N T S

COMPONENTS

OF T H E E R Y T H R O C Y T E

As d e t e r m i n e d by different methods [12], the erythrocytes are estimated to contain approximately 2 × 100 ABH-determinants per cell. The p r o p o r t i o n s occurring in the different molecular forms can be determined in different ways. The n u m b e r s as calculated f r o m the amounts of determinants in different isolated c o m p o n e n t s are only rough approximations, due to the variable yields and the difficulties in recalculations [12]. Another a p p r o a c h is the incorporation of radioactive N-acetylgalactosamine by blood-group A dependent transferase to O erythrocytes, followed by isolation of the individual c o m p o n e n t s [18-20]. A third a p p r o a c h is to m e a s u r e the a m o u n t of lectin b o u n d to the electrophoretically separated c o m p o n e n t s (Fig. 4) [12]. The a p p r o x i m a t e p r o p o r t i o n s of the ABH determinants as measured by different m e t h o d s are s u m m a r i z e d in Table I. Although each m e t h o d has its limitations [12], the values reveal a fairly similar pattern. It therefore seems that the m a j o r i t y of the blood-group activity can

550

FINNE

Y. and coll.

TABLE I Distribution of blood-group ABH determinants among different components of the human erythrocyte membrane. COMPONENT

CHEMICAL AMOUNT

TRANSFERASE METHOD

5 25-40

of total 5 10-20

LECTIN BINDING

p. c e n t

Small glycosphingolipids . . . . . . . . . . . . . . Polyglycosyl ceramides . . . . . . . . . . . . . . . . Alkali-labile chains of glycoproteins (PAS-bands) . . . . . . . . . . . . . . . . . . . . . . . Alkali-stable (polyglycosyl) chains of glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . Band 3 . . . . . . . . . . . . . . . . . . . . . . . . . . Band 4.5 upper part . . . . . . . . . . . Band 4.5 lower part . . . . . . . . . . . .

ND 50-75 ND ND ND

aND 15

5-15

25

60-75 ND ND ND

65 25 10 25

aND = not determined. The chemical amounts for the isolated components are rough estimates and are based on the values summarized in ref. [12]. Values for the transferase method are from refs [18-20] and those for lectin binding from ref. [12]. b e a s c r i b e d to t h e p o l y g l y c o s y l c h a i n s , w h i c h o c c u r b o u n d to d i f f e r e n t p r o t e i n s a n d lipids.

CONCLUSIONS I t s e e m s at p r e s e n t e v i d e n t t h a t t h e b l o o d - g r o u p A B H d e t e r m i n a n t s o c c u r o n s e v e r a l d i f f e r e n t classes of g l y c o c o n j u g a t e s o n t h e h u m a n erythrocyte membrane. T h i s w o u l d i n d i c a t e t h a t t h e glycosyltransf e r a s e s c a t a l y z i n g t h e b i o s y n t h e s i s of t h e s e s t r u c t u r e s o n l y recognize t h e specific s t r u c t u r e of t h e a c c e p t o r o l i g o s a c c h a r i d e , b u t n o t of the c a r r i e r m o l e c u l e , t h a t is, t h e p r o t e i n or l i p i d p a r t . I t is p o s s i b l e that t h i s c o u l d also b e t h e case for t h e p r o t e i n - a n d l i p i d - b o u n d carboh y d r a t e s i n g e n e r a l , as i n d i c a t e d b y t h e h i g h d e g r e e of s i m i l a r i t y of t h e t e r m i n a l s u g a r s e q u e n c e s i n t h e s e t w o classes of c o m p o u n d s [21]. T h e s t u d i e s o n t h e b l o o d - g r o u p ABH a n t i g e n s also give a n i n d i c a t i o n o n w h a t t y p e of a p p r o a c h e s s h o u l d b e u s e d , w h e n b i o l o g i c a l activities d e t e r m i n e d by carbohydrate structures are investigated. S i n c e the p r o t e i n - o r l i p i d - c a r r i e r m a y b e of m i n o r i m p o r t a n c e f o r t h e biological a c t i v i t y of t h e c a r b o h y d r a t e p a r t , t h e a p p r o a c h u s i n g t h e i s o l a t i o n of a single g l y c o p r o t e i n o r g l y c o l i p i d is u n l i k e l y to b e v e r y fruitful, a n d m a y l e a d to c o n f u s i o n . I n s t e a d m e t h o d s a r e n e e d e d w h i c h take i n t o c o n s i d e r a t i o n t h e specific s t r u c t u r e of t h e c a r b o h y d r a t e chains, s u c h as t h e u s e of l e c t i n s , g l y c o s y l t r a n s f e r a s e s a n d o t h e r c a r b o h y d r a t e s p e c i f i c m o l e c u l e s , as w e l l as t h e s t u d y of o l i g o s a c c h a r i d e s a n d glycop e p t i d e s [81 r e l e a s e d f r o m t h e i r p r o t e i n - c a r r i e r s .

551

BLOOD GROUP ABH ANTIGENS

ABSTRACT The o l i g o s a c c h a r i d e s t r u c t u r e s s p e c i f y i n g t h e b l o o d - g r o u p A B H d e t e r m i n a n t s o c c u r in t h e h u m a n e r y t h r o c y t e m e m b r a n e in d i f f e r e n t classes of c o m p o u n d s . T h e m a j o r i t y o c c u r in a n o v e l c l a s s o f c o m p l e x carbohydrate chains called the polyglycosyl chains. They are bound by a n a l k a l i - s t a b l e b o n d to g l y c o p r o t e i n s ( b a n d 3, b a n d 4.5) a n d o c c u r also in g l y c o l i p i d s . C o n v e n t i o n a l g l y c o s p h i n g o l i p i d s as w e l l as a l k a l i labile c a r b o y h y d r a t e c h a i n s o f g l y c o p r o t e i n s (in t h e P A S - b a n d s ) a r e also c a r r i e r s of t h e b l o o d - g r o u p d e t e r m i n a n t s . RESUME Les o l i g o s a c c h a r i d e s q u i d 6 t e r m i n e n t chez les h u m a i n s la s p 6 c i f i c i t 6 ABH des g r o u p e s s a n g u i n s f o n t p a r t i e d e d i f f 6 r e n t s c o m p o s a n t s m e m branaires des hdmaties. La majorit6 d'entre eux correspond gun nouveau type d e c h a i n e s p o l y s a c c h a r i d e s , les c h a i n e s p o l y g l y c o s i d i q u e s . Celles-ci sont u n i e s ~ d i f f 6 r e n t e s g l y c o p r o t 6 i n e s ( b a n d e 3, b a n d e 4.5) p a r u n e liaison s t a b l e h l ' h y d r o l y s e a l c a l i n e , a i n s i qu'~t d e s g l y c o l i p i d e s . La sp6cificit4 d e s g r o u p e s s a n g u i n s e s t d ' a u t r e p a r t a u s s i d 4 t e r m i n 6 e p a r les oses d e s s p h i n g o s i d o l i p i d e s c o n v e n t i o n n e l s , et p a r les c h a i n e s oligos a c c h a r i d e s li4es ~ d e s g l y c o p r o t d i n e s ( c e l l e s d e s b a n d e s PAS) p a r d e s liaisons s e n s i b l e s g l ' h y d r o l y s e a l c a l i n e . Request r e p r i n t s f r o m : J u k k a FINNE, D e p a r t m e n t of Medical Chemistry University of HELSlNKI (Finland). ACKNOWLEDGEMENTS. - - O u r w o r k has been s u p p o r t e d by grants f r o m the Sigrid Jusdlius Foundation, Finland. P a r t of the w o r k was c a r r i e d out in the l a b o r a t o r y of Dr. MM. BURGER at the Biocenter of the University of Basel, and was s u p p o r t e d by a g r a n t (3.720-76) f r o m the Swiss National Foundation.

REFERENCES

[1] WATKIRS W.M. - - Blood-group substances. Science, 152, 172, 1966. [2] HAKOMORI S- - - Glycolipids of animal cell m e m b r a n e s . In : G.O. Aspinall (ed.), MTP I n t e r n a t i o n a l Review of Science. Organic Chemistry Series two, 7, 223, 1976. [3] KOSClELAK J., MILLER-PODRAZA H., KRAUZE R. and PIASEK A. - - Isolation and c h a r a c t e r i z a t i o n of poly(glycosyl)-ceramides (megaloglycolipids) with A, H and I blood-group activities_ Eur. J_ Biochem., 71, 9, 1976. [41 DEJTER-JUszYNsKI M., HARPAZ N., FLOWERS H.M. and SHARON N. - - Bloodgroup ABH-specific macroglycolipids of h u m a n erythrocytes : isolation in high yield from a crude glycoprotein fraction. Eur. J. Biochem., 83, 363, 1978.

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[5] MARCHESI V.T. and ANDREWS, E.P. - - Glycoproteins : isolation f r o m cell m e m b r a n e s with l it h i u m diiodosalicylate. Science, 174, 1247, 1971. [6] HAMAGUCHI H. and CLEVE H. - - Solubilization of h u m a n erythrocyte m e m b r a n e glycoproteins and s e p a r a t i o n of the MN glycoprotein f r o m a glycoprotein w i t h I, S, and A activity. Biochim. Biophys. Acta, 278, 271, 1972. [ 7 ] TAKASAKIS. and KOBATAA. - - Chemical c h a r a c t e r i z a t i o n and distribution of ABO blood-group active glycoprotein in h u m a n e r y t h r o c y t e membrane. J. Biol. Chem., 251, 3610, 1976. [8] FINNE J., KRUSlUS T. and JARNEFELT J. - - F r a c t i o n a t i o n of glycopeptides. In : A. V a r m a v u o r i (ed.), 27th I n t e r n a t i o n a l Congress of Pure and Applied Chemistry, p. 147, P e r g a m o n Press, Oxford and New York, 1980. [9] FINNE J., KRUSlUS T., RAUVALAH., KEKOMAKI R. and MYLLYLA G. - - Alkalistable blood-group A- and B-active poly(glycosyl)-peptides f r o m h u m an e r y t h r o c y t e m e m b r a n e . F E B S Lett., 89, 111, 1978. [10] KRUSlUS T., FINNE J. and RAUVALA H. - - The poly(glycosyl) chains of glycoproteins. Characterization of a novel type of glycoprotein saccharides f r o m h u m a n e r y t h r o c y t e membrane_ Eur. J. Biochem., 92, 289, 1978. [111 JARNEFELT J . , R U S H J . , LI Y.-T. and LAINE R.A. - - Erythroglycan, a high m o l e c u l a r weight glycopeptide w i t h the rep eat i n g s t r u c t u r e [galactosyl(1 ~4)-2-deoxy-2-acetamido-glucosyl (1 ~ 3 ) c o m p r i s i n g m o r e than one t h i r d of the protein-bound c a r b o h y d r a t e of h u m a n e r y t h r o c y t e stroma. J. Biol. Chem., 253, 8006, 1978. [12] FINNE J. - - I d e n t i f i c a t i o n of the blood-group ABH-active glycoprotein c o m p o n e n t s of h u m a n e r y t h r o c y t e m e m b r a n e . Eur. J. Biochem., 104, 181, 1980. [13] MURPHY L.A. and GOLDSTEIN I.J. - - Physical-chemical c h a r a c t e r i z a t i o n and carbohydrate-binding activity of the A and B subunits of the Bandeiraea simpliciiolia I isolectins_ Biochemistry, 18, 4999, 1979. [14] KABATE.A. - - Dimensions and specificities of recognition sites on lectins and antibodies. J. Supramol. Struct., 8, 79, 1978_ [15] TAKASAKIS., YAMASHITAK. and KOBATAA. - - The sugar chain structures of ABO blood-group active glycoproteins obtained f r o m h u m a n erythrocyte m e m b r a n e . J. Biol. Chem., 253, 6086, 1978. [16] FUKUDA M.N., FUKUDA M. and HAKOMORI S. - - Cell surface modification by endo-~-galactosidase. Change of blood group activities and release of oligosaccharides f r o m glycoproteins and glycosphingolipids of h u m a n erythrocytes. 1. Biol. Chem., 254, 5458, 1979_ [17] MUELLER T . J . , LI Y.-T. and MORRISON M. - - E f f e c t of endo-~-galactosidase on intact erythrocytes. J. Biol. Chem., 254, 8103, 1979. [ 1 8 ] SCHWYZER M. and HILL R.L. - - Porcine A blood group-specific Nacet y l g al act o s a m in y l tr a n s f e r a s e . II. E n z y m a t i c Properties. J. Biol. Chem., 252, 2346, 1977. [19] SCHENKEL-BRUNNERH. - - Blood-group ABH antigens of h u m a n erythrocytes. Quantitative studies on the distribution of H antigenic sites among different classes of m e m b r a n e components. Eur. J. Biochem., 104, 529, 1980. [20] WlLZYNSKA Z., MILLER-PODRAZA H. and KOSCIELAK J. - - The contribution of d i f f eren t glycoconjugates to the total At~.H blood group activity of h u m a n erythrocytes. F E B S Lett., 112, 277, 1980_ [21] RAUVALAH. and FINNE J. - - S t r u c t u r a l similarity of the t e r m i n a l carboh y d r a t e sequences of glycoproteins and glycolipids. F E B S Lett., 97, 1, 1979.