Carbohydrate Antigens - American Chemical Society

Chapter 4 Human Fucosyltransferases

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 15, 2016 | http://pubs.acs.org Publication Date: December 17, 1993 | doi: 10.1021/bk-1993-0519.ch004

Involved in the Biosynthesis of X (Gal-β-1-4[Fuc-α1-3]GlcNAc) and Sialyl-X (NeuAC-α-2-3Gal-β-1-4[Fuc-α1-3]GlcNAc) Antigenic Determinants 1

2

3

Winifred M. Watkins , Patricia O. Skacel , and Philip H. Johnson 1

Department of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 ONN, England Department of Haematology, Northwick Park Hospital, Watford Road, Harrow, Middlesex HA1 3UJ, England Medical Research Council Human Biochemical Genetics Unit, University College, London NW1 2HE, England

2

3

The X structure (Gal-β-1-4[Fuc-α-1-3]GlcNAc) occurs in glycoproteins, glycolipids, and free oligosaccharides and may be exposed as a terminal non-reducing end-group or masked by substitution with other sugars. The presence of s i a l i c acid in α-2,3-linkage to the term inal β-galactosyl residue gives the sialyl-X determinant (NeuAc-α-2-3Gal-β-1-4-[Fuc-α-1-3]GlcNAc). Both structures have been identified as human tumour markers and as ligands in cellular adhesion reactions mediated by endo genous lectins (LEC-CAMS). X structures are biosynthesised by a family of GDP-fucose:-N -acetyl-D-glucosaminide α-3-L-fucosyltransfer ases. The obligatory pathway for biosynthesis of sialyl-X i s first the formation of NeuAc α2-3-Gal-β-1-4GlcNAc, followed by the addition of the fucosyl residue to the N-acetylglucosamine unit. Not all α-3-fucosyltransferase species that synthesise X determinants can add fucose to the GlcNAc residue in NeuAcα-23Gal-β-1-4-GlcNAc to form sialyl-X determin ants. In myeloid tissues species differing in their activities with sialylated acceptors occur at different stages of maturation; these variants appear to be encoded by different genes from the α-3-fucosyltransferases ex pressed in other tissues. 0097-6156/93/0519-0034$08.50/0 © 1993 American Chemical Society

Garegg and Lindberg; Carbohydrate Antigens ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


4. WATKINS ET AL.

Human Fucosyltransferases

35

The c a r b o h y d r a t e m o i e t i e s o f g l y c o p r o t e i n s , g l y c o s p h i n g o l i p i d s and g l y c o s a m i n o g l y c a n s a r e i n v o l v e d i n many c e l l s u r f a c e phenomena, i n c l u d i n g a n t i b o d y and l e c t i n b i n d i n g , cellular adhesion and recognition. Changes that take p l a c e i n the surface carbohydrate s t r u c t u r e s are thought t o p l a y an e s s e n t i a l p a r t i n n o r m a l c e l l u l a r d i f f e r e n t i a t i o n and to i n f l u e n c e the m e t a s t a t i c p o t e n t i a l o f cancer c e l l s . The p o s s i b i l i t y o f s t u d y i n g t h e s e changes h a s b e e n greatly facilitated by the advent of the hybridoma t e c h n i q u e (1^) w h i c h h a s a l l o w e d t h e p r o d u c t i o n o f a r a n g e of h i g h l y s p e c i f i c monoclonal a n t i b o d i e s f o r the d e t e c t i o n o f c l o s e l y r e l a t e d c a r b o h y d r a t e s t r u c t u r e s ( 2 ^ ) . Of equal importance f o r an u n d e r s t a n d i n g o f the b i o c h e m i c a l and g e n e t i c m e c h a n i s m s u n d e r l y i n g t h e c h a n g i n g p a t t e r n o f c e l l s u r f a c e s t r u c t u r e s has been the c h a r a c t e r i s a t i o n o f t h e g l y c o s y l t r a n s f e r a s e e n z y m e s ( r e v i e w e d i n r e f s 4^ & 5) responsible for the biosynthesis o f the determinants. Assay of these glycosyltransferases enables attempts to b e made t o c o r r e l a t e s u r f a c e a n t i g e n i c c h a n g e s w i t h l o s s or o v e r p r o d u c t i o n o f the enzymes, and the r e c e n t success in cloning g l y c o s y l t r a n s f erase genes (6."".8) i s already p r o v i d i n g v a l u a b l e probes f o r f u r t h e r study o f the geneti c r e g u l a t i o n and t i s s u e s p e c i f i c e x p r e s s i o n o f the enzymes and a n t i g e n s . L-Fucose and s i a l i c acid (N-acetylneuraminic acid) are sugars involved i n the terminal glycosylation of oligosaccharide chains of glycoconjugates and as such p l a y a major r o l e i n the a n t i g e n i c p r o p e r t i e s o f cell s u r f a c e s and i n the c h a n g i n g c a r b o h y d r a t e p r o f i l e s t h a t o c c u r o n c e l l m a t u r a t i o n and i n m a l i g n a n c y . T h i s communi c a t i o n w i l l d i s c u s s 1) some o f t h e a n t i g e n i c structures i n v o l v i n g fucose and s i a l i c a c i d , w i t h an emphasis on X a n d s i a l y l - X ( F i g u r e 1 ) , 2) t h e r o l e s o f t h e s e structures as a n t i g e n i c d e t e r m i n a n t s , tumour markers and l i g a n d s f o r a d h e s i o n m o l e c u l e s a n d 3) t h e p r o p e r t i e s a n d i n t e r r e l a t i o n s h i p s o f the f u c o s y l t r a n s f e r a s e s responsible for the b i o s y n t h e s i s o f X and s i a l y l - X .

F u c o s y l - l i n k a g e s i n Human Glycoconjugates Four different types o f f u c o s y l - l i n k a g e s are commonly f o u n d i n human g l y c o c o n j u g a t e s ( F i g u r e 2 ) . The f i r s t two to be c l e a r l y a s s o c i a t e d w i t h a n t i g e n i c d e t e r m i n a n t s were fucose l i n k e d a-1,4 to a subterminal N-acetylglucosami n y l r e s i d u e i n a Type 1 (Gal-j(3-l-3GlcNAc) c h a i n t o g i v e the L e a n t i g e n i c determinant ( £ ) and fucose l i n k e d a 1,2 t o a j 8 - g a l a c t o s y l r e s i d u e i n e i t h e r a Type 1 o r a Type 2 (Gal-jg-l-4GI cNAc) o l i g o s a c c h a r i d e c h a i n e n d i n g , to g i v e blood group H a n t i g e n i c s t r u c t u r e s (10). Subsequently the presence o f both a - l , 2 - l i n k e d and 1 , 4 - l i n k e d fucose i n a Type 1 c h a i n s t r u c t u r e (Fuc-a-12 G a l - / 3 - l - 3 [ F u c - a - l - 4 ] G l c N A c ) was f o u n d t o be responsible for Le antigenic specificity (11.). C h a r a c t e r i s a t i o n o f t h i s d e t e r m i n a n t showed t h a t the a d d i t i o n o f a monosacch a r i d e s u b s t i t u e n t to a sugar residue adjacent to a determinant structure i n an o l i g o s a c c h a r i d e c h a i n , whether d i s t a l or p r o x i m a l to the f i r s t s u b s t i t u e n t , masks the a

j

b


36

CARBOHYDRATE ANTIGENS


STRUCTURE

REFERENCE

X-determinant Gal jSl-4

N

G l c N A c - β-R Fuc

14,16

a 1-3'

Sialyl-X

determinant

NeuAc a 2 - 3 G a l β

1-4 ^GlcNAc-β-R V

23-27

F u c a 1-3

VIM

2

determinant

N e u A c α 2-3Gal β l-4GlcNAc /3l-3Gal

01-4 x

Fuc

Dimeric-X

a 1-3

GlcNAc-β-R '

28

determinant

Gal β 1-4. GlcNAc β l - 3 G a l β

1-4.

Fucα

1-3'

F u c a 1-3'

GlcNAc-β-R

30

Figure 1. S t r u c t u r e s o f X , S i a l y l - X , VIM 2 and D i m e r i c - X d e t e r m i n a n t s . A l l sugars are i n the pyranose form. A b b r e v i a t i o n s : - G a l , D - g a l a c t o s e ; Fuc, L - f u c o s e ; GlcNAc, N - a c e t y l - D - g l u c o s a m i n e ; NeuAc, N - a c e t y l n e u r aminic a c i d R, remainder o f molecule.


4. WATKINS ET AL.



Linkage formed by Fucosyltransferase

Acceptor Substrate

37

Structure Formed

Fuc- a-2-

G a l - 0 -R

Fuc-a-l-2Gal- β -R

Fuc-a-3-

Gal-jS-l-4GlcNAc-R (Type2)

Gal-jS-1-4 GlcNAc-R Fuc-a-1-3

Fuc-a-3-

Gal-jfrl-4Glc (Lactose)

Gal-/3-l-4

x

Glc Fucα 1-3

Fuc-α -4-

Gal-|3-l-3GlcNAc-R (Type 1)

Gal-^-l-3 ν GlcNAc-R Fuc-a-1-4

Fuc-a -6-

R. . .GlcNAc-|8-l-4GlcNAc-ASN

y

R. . .GlcNAc-/3-l-4GlcNAc-ASN

6 :

ι 1

Fuc

F i g u r e 2. Fucosyl l i n k a g e s commonly found i n human glycoconjugates. A b b r e v i a t i o n s : - G l c , D-glucose; ASN, asparagine; others as i n Figure 1.


·

38


o r i g i n a l s p e c i f i c i t y and g i v e s r i s e to a s t r u c t u r e that h a s i t s own d i s t i n c t i v e a n t i g e n i c properties. I n g e n e r a l , Type 2 H d e t e r m i n a n t s o c c u r as t e r m i n a l structures i n g l y c o s p h i n g o l i p i d s and in glycoproteins w i t h b o t h O - l i n k e d and N - l i n k e d o l i g o s a c c h a r i d e chains and Type 1 determinants (H, L e and L e ) in glyco s p h i n g o l i p i d s and i n g l y c o p r o t e i n s w i t h £ - l i n k e d chains (4). No a n t i g e n i c a c t i v i t y h a s b e e n a s s o c i a t e d w i t h f u c o s e linked a-1,6 to N-acetylglucosamine (Figure 2 ) . This l i n k a g e appears to occur e x c l u s i v e l y i n the core r e g i o n on the N - a c e t y l g l u c o s a m i n e r e s i d u e a t t a c h e d t o asparagine i n the peptide backbone o f g l y c o p r o t e i n s w i t h N-linked chains (12). F u c o s e l i n k e d α - 1 , 3 t o N - a c e t y l g l u c o s a m i n e was f i r s t reported i n o l i g o s a c c h a r i d e s obtained by degradation of ^ - l i n k e d chains i n blood-group-active ovarian cyst glyco p r o t e i n s . L l o y d et a l (13) o b t a i n e d fragments from A - and Η-active glycoproteins that contained internal linkages of fucose joined a-1,3 t o N - a c e t y l g l u c o s a m i n e . The t r i saccharide sequence, G a l - j g - l ^ [ F u c a - 1 - 3 ] G l c N A c , was f i r s t i s o l a t e d i n 1968 b y M a r r e t a l . (14) as a t e r m i n a l n o n reducing structure from a g l y c o p r o t e i n p r e p a r a t i o n ob tained from the cyst fluid o f an L e - p o s i t i v e donor. S i n c e o v a r i a n c y s t g l y c o p r o t e i n s have b o t h Type 1 and T y p e 2 o l i g o s a c c h a r i d e c h a i n s (1J> ) , p r e p a r a t i o n s obtained f r o m L e - p o s i t i v e d o n o r s , who a r e A B H - n o n - s e c r e t o r s a n d hence l a c k t e r m i n a l A - , B - , or Η - a c t i v e s t r u c t u r e s , can yield oligosaccharide fragments i n which the terminal n o n - r e d u c i n g end groups o f Type 1 c h a i n s h a v e , as the only substituent, fucose i n a - 1 , 4 l i n k a g e to the subter minal N-acetylglucosamine (Le determinant) and Type 2 c h a i n s h a v e , a l s o as the o n l y s u b s t i t u e n t , fucose l i n k e d α-1,3 to N-acetylglucosamine (X determinant).The i s o l a t e d t r i s a c c h a r i d e was r e c o g n i s e d as a n i s o m e r o f L e t h a t was w i t h o u t L e i m m u n o l o g i c a l a c t i v i t y ( 1 4 ) . T h e name X - h a p t e n w a s s u b s e q u e n t l y a p p l i e d to the Type 2 t r i s a c c h a r i d e w i t h a - 3 - l i n k e d fucose when a glycosphingolipid with this terminal non-reducing seq u e n c e w a s i s o l a t e d f r o m h u m a n a d e n o c a r c i n o m a t i s s u e (160 · The l i n k a g e i s now k n o w n t o o c c u r i n a v a r i e t y o f g l y c o p r o t e i n s w i t h b o t h 0- a n d N - l i n k e d o l i g o s a c c h a r i d e c h a i n s a n d i n f r e e l a c t o s e - b a s e d o l i g o s a c c h a r i d e s i n human m i l k ( 1 7 ) a n d u r i n e (1.8 ) . I n p o l y - N - a c e t y l l a c t o s a m i n e c h a i n s i n g l y c o p r o t e i n s a n d g l y c o l i p i c t s f u c o s e r e s i d u e s may a l s o be a t t a c h e d to the £ - 3 - p o s i t i o n of internal N-acetyl glucosamine residues (Dimeric-X, Figure 1) (_19). The l a c t o s e - b a s e d o l i g o s a c c h a r i d e s can a l s o have fucose link ed a - 1 - 3 t o t h e g l u c o s e r e s i d u e i n t h e l a c t o s e ( G a l j8l4 G l c ) s t r u c t u r e (Uj-18) ( F i g u r e 2 ) . Partial characterisation o f a human a n t i b o d y that agglutinated erythrocytes of individuals grouped as "Le(a-b-), non-secretors of ABH" suggested that this a n t i b o d y , d e s i g n a t e d as a n t i - L e , m i g h t be d e t e c t i n g the X - h a p t e n i c s e q u e n c e ( 2 0 ) . No o t h e r naturally occurring


a

b

a

a

a

a

a

c



4. WATKINS ET AL.


39

human a n t i b o d i e s r e a c t i n g w i t h t h i s s t r u c t u r e appear to have been r e p o r t e d . With the i n t r o d u c t i o n o f the h y b r i d oma technique for the production of monoclonal anti b o d i e s , however, the Gal-|3-l-4[Fuc-a-1-3]GlcNAc s t r u c t u r e was f o u n d t o b e s t r o n g l y i m m u n o g e n i c i n m i c e a n d a n t i b o d i e s r a i s e d a g a i n s t a v a r i e t y o f c e l l l i n e s , as w e l l as t h o s e r a i s e d a g a i n s t b o t h n o r m a l and tumour t i s s u e s , were subsequently found to recognise t h i s t r i s a c c h a r i d e seq u e n c e ( 2 - 3 , 2 1 ) . Among t h o s e f r e q u e n t l y r e f e r r e d t o i n t h e l i t e r a t u r e are an a n t i b o d y d i r e c t e d to a stage s p e c i f i c e m b r y o n i c a n t i g e n i n t h e mouse ( S S E A 1, r e f . 2 2 ) and a g r o u p o f h u m a n l e u k o c y t e a n t i b o d i e s c a l l e d CD 1 5 " ~ ( C l u s t e r of Differentiation 15; International Workshop on Differentiation Antigens, 1987). The symbols Le or L e w i s - x are a l s o used f o r the t r i s a c c h a r i d e sequence and the difucosyl structure ( F u c - α - l - 2 G a l -j8 1 - 4 [ F u c - a - 1 - 3 ] G l c N A c ) , the Type 2 c h a i n i s o m e r o f L e ° , i s known as Y , Le > or L e w i s - y . x

v

Hybrid S t r u c t u r e s c o n t a i n i n g Fucose and S i a l i c

acid

The Type 1 and Type 2 s t r u c t u r e s i n g l y c o p r o t e i n s and glycolipids are substrates not only for the fucosyl t r a n s f e r a s e s but a l s o f o r the enzymes t h a t t r a n s f e r N a c e t y l n e u r a m i n i c a c i d ( s i a l i c a c i d ) to the t e r m i n a l β g a l a c t o s y l r e s i d u e s t o f o r m NeuAc 2 - 3 G a l and NeuAc 2-6G a l l i n k a g e s (J5). G l y c o c o n j u g a t e m o l e c u l e s b e a r i n g s i a l i c a c i d and fucose j o i n e d to adjacent sugar r e s i d u e s ( F i g u r e 1) h a v e b e e n i s o l a t e d f r o m b o t h n o r m a l human t i s s u e s a n d f r o m t u m o u r s ( 2 3 - 2 7 ) . When t h e s i a l i c a c i d i s l i n k e d a 2,3 on the t e r m i n a l β-galactosyl r e s i d u e the structure formed on Type 1 c h a i n s is sialyl-Le and on Type 2 chains i s s i a l y l - X (or s i a l y l - L e ) . Monoclonal antibodies s p e c i f i c f o r these h y b r i d s t r u c t u r e s have been r a i s e d by i m m u n i s a t i o n o f m i c e w i t h c a n c e r c e l l membranes o r w i t h purified glycolipids antigens coated onto bacteria ( 2 2 , 2 2 ) . The p r e s e n c e o f the s i a l i c a c i d r e s i d u e masks the s p e c i f i c i t y o f the L e or X s t r u c t u r e s i n t h e same way a s t h e p r e s e n c e o f a f u c o s y l r e s i d u e o n t h e t e r m i n a l g a l a c t o s e masks the s p e c i f i c i t y o f L e i n Le" structures and X i n Y s t r u c t u r e s . An a d d i t i o n a l s t r u c t u r e carrying a terminal a-2,3linked sialic acid and a fucose on an internal Na c e t y l g l u c o s a m i n e residue o f a p o l y - N - a c e t y l l a c t o s a m i n e a

x

a

a

chain ( F i g u r e by

1) i s

recognised a s

the monoclonal antibody V I M 2

an Independent

antigen

(2j8).

X and S i a l y l - X as Tumour-Associated and D i f f e r e n t i a t i o n Antigens A l t h o u g h X and s i a l y l - X s t r u c t u r e s have been i d e n t i f i e d i n g l y c o c o n j u g a t e s f r o m n o r m a l human t i s s u e s b o t h o c c u r i n h i g h e r d e n s i t y as s u r f a c e components i n c e r t a i n tumour tissues. G l y c o s p h i n g o l i p i d s c a r r y i n g the X determinant


40


w e r e f o u n d t o a c c u m u l a t e i n human l u n g , g a s t r i c a n d c o l onic cancers (16>,2£) and compounds w i t h poly-N-acetyllactosamine chains have been isolated in wTTich two ( F i g u r e 1) and t h r e e fucose r e s i d u e s are j o i n e d i n ot^ 3-linkage to successive i n t e r n a l N-acetylglucosamine resi d u e s ( 3 0 ) · I n a d d i t i o n many human c a n c e r s , p a r t i c u l a r l y adenocarcinomas, have been c h a r a c t e r i s e d by the presence


of

the s i a l y l - X

antigen i n organs i n w h i c h

it is

either

n o t n o r m a l l y e x p r e s s e d , o r o n l y t o a much l e s s e r extent ( 2 5 , 3 0 - 3 1 ) . The r o l e o f X and s i a l y l - X as tumour m a r k e r s has suggested the use o f m o n o c l o n a l a n t i b o d i e s d i r e c t e d towards these determinants as t h e r a p e u t i c reagents for d r u g t a r g e t i n g and tumour s u p p r e s s i o n (29)* I n human h a e m o p o i e t i c t i s s u e X a n t i g e n i c e x p r e s s i o n a p p e a r s t o be d e v e l o p m e n t a l l y r e g u l a t e d s i n c e i t i s found on m a t u r e n e u t r o p h i l s and on a l l m y e l o i d c e l l s f r o m t h e p r o m y e l o c y t i c s t a g e onwards but i s absent from e a r l i e r p r e c u r s o r s ( 3 2 - 3 3 ) . The X s t r u c t u r e was f i r s t r e c o g n i s e d as a d e v e l o p m e n t a l l y r e g u l a t e d a n t i g e n i n t h e mouse i n a s much as a m o n o c l o n a l a n t i b o d y d i r e c t e d t o t h e undiffere n t i a t e d t e r a t o c a r c i n o m a c e l l l i n e F9 ( a n t i - S S E A 1, ref. 22), w h i c h was shown t o be r e a c t i n g w i t h t h e X s e q u e n c e (34,), was f o u n d t o be m a x i m a l l y e x p r e s s e d a t t h e m o r u l a to e a r l y b l a s t o c y s t s t a g e s and to d e c l i n e at the later s t a g e s o f d e v e l o p m e n t . I n human t i s s u e s X a n d D i m e r i c - X are expressed i n g a s t r o i n t e s t i n a l t i s s u e e a r l y i n embryog e n i s i s but v i r t u a l l y d i s a p p e a r from the mucosa o f newborn infants and a d u l t s , only to reappear in gastroi n t e s t i n a l tumours ( 3 0 ) .

The Role o f X and S i a l y l - X as Ligands f o r C e l l Molecules

Adhesion

The i n t e r e s t g e n e r a t e d b y t h e f i n d i n g t h a t X a n d s i a l y l - X a r e tumour and d i f f e r e n t i a t i o n markers has r e c e n t l y been far exceeded by interest in the finding that these c a r b o h y d r a t e s t r u c t u r e s p o s s i b l y f u n c t i o n as l i g a n d s f o r the f a m i l y o f a d h e s i o n m o l e c u l e s known as the LEC-CAMS (or Selectins). Adhesion of c i r c u l a t i n g leukocytes to vascular endothelial c e l l s i s a key step i n inflammatory responses and the LEC-CAMs have been i m p l i c a t e d i n the interaction of leukocytes with platelets or vascular e n d o t h e l i u m ( 3 5 - 3 6 ) . These p r o t e i n s are a unique family of cell adhesion molecules c h a r a c t e r i s e d by the juxtap o s i t i o n o f an N - t e r m i n a l l e c t i n domain, an epidermal growth f a c t o r domain, and v a r i a b l e numbers o f complement regulatory p r o t e i n - l i k e repeating units (reviewed i n refs 37 & 3 8 . ) · T h e m i n i m a l o l i g o s a c c h a r i d e b o u n d b y L E C - C A M 1 , which is c o n s t i t u t i v e l y expressed on the majority of leukocytes and f a c i l i t a t e s binding of these cells to e n d o t h e l i u m d u r i n g lymphocyte r e c i r c u l a t i o n , has not yet been c l e a r l y d e f i n e d but s i a l i c a c i d or other charged groups have been i m p l i c a t e d (3jJ). The l i g a n d most f i r m l y e s t a b l i s h e d a p p e a r s t o be



4. WATKINS ET AL.


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t h a t r e c o g n i s e d b y L E C - C A M 2 ( a l s o k n o w n a s ELAM 1 ) w h i c h i s t r a n s i e n t l y expressed by e n d o t h e l i a l c e l l s i n response to inflammatory agents. S e v e r a l groups have concluded that this adhesion molecule recognises the sialyl-X structure ( 4 0 - 4 2 ) . An a l t e r n a t i v e suggestion (43) that t h i s LEC-CAM r e c o g n i s e s the VIM 2 d e t e r m i n a n t ( F i g u r e 1 ) , has not been c o n f i r m e d by recent work ( 4 4 ) . L E C - C A M 3 t h e t h i r d member o f t h i s " F a m i l y ( a l s o k n o w n a s P A D G E M , GMP 1 4 0 o r C D 6 2 ) i s f o u n d i n p l a t e l e t s and e n d o t h e l i a l c e l l s and b i n d s to n e u t r o p h i l s and monocytes at the s i t e o f t i s s u e i n j u r y . A n t i b o d y b l o c k i n g e x p e r i ments and the use o f s o l u b l e i n h i b i t o r s s t r o n g l y indi cated that the X - h a p t e n i c s t r u c t u r e , or a c l o s e l y r e l a t e d o l i g o s a c c h a r i d e sequence, i s i n v o l v e d i n the b i n d i n g o f this adhesion molecule (45). The demonstration of the role of sialyl-X and related carbohydrate structures as l i g a n d s f o r adhesion m o l e c u l e s h a s s u g g e s t e d new p o s s i b i l i t i e s f o r t h e d e v e l opment o f t h e r a p e u t i c a g e n t s f o r t r e a t m e n t o f i n f l a m m a t ory conditions and appears at last to have brought glycoconjugates into the forefront o f b i o c h e m i c a l and biomedical thought (37-38, 44,46).

B i o s y n t h e s i s o f X and S i a l y l - X S t r u c t u r e s

Is

the X

Structure

Biosynthesised

by

the Lewis

Gene-

Associated α-3/4-Fucosyltransferase? GDP-L-fucose: galactosyl-/3-l-4-N-acetyl- #-D-glucosaminide α-3-fucosyltransferases a b l e to t r a n s f e r fucose to the 0 - 3 - p o s i t i o n o f the p e n u l t i m a t e N - a c e t y l g l u c o s a m i n e i n Type 2 s t r u c t u r e s to form the X - d e t e r m i n a n t were e a r l y d e t e c t e d in human milk, submaxillary glands, stomach mucosa and plasma (47-49). Although a l l the t i s s u e s e x p r e s s i n g the L e w i s Le gene a s s o c i a t e d G D P - L - f u c o s e : g a l a c t o s y l j g - l - 3 - N a c e t y l — β-D-glucosaminide α-4-fucosyltransferase also have 3 - f u c o s y l t r a n s f e r a s e a c t i v i t y not a l l the tissues with α - 3 - a c t i v i t y have a - 4 - a c t i v i t y . This fact, to gether w i t h the finding that 3-fucosyltransferase act ivity is expressed in tissues of individuals of the genotype L e ( a - b - ) , i n d i c a t e d that the α-3-enzyme is en c o d e d b y a g e n e i n d e p e n d e n t o f t h e L e w i s g e n e (4^). The d i f f e r e n c e s i n t i s s u e s p e c i f i c e x p r e s s i o n o f the a - 3 - and the α - 4 - e n z y m e s , together with the known f r e q u e n c y of i n d i v i d u a l s homozygous f o r the Le gene ( ^ 0 ) , a l s o s u g g e s t e d that the 3 - f u c o s y l t r a n s f e r a s e i s not the product o f t h e s e c o n d JLe a l l e l e a t t h e L e w i s l o c u s ( 4 ) . Subsequently e x a m i n a t i o n o f s a l i v a from i n d i v i d u a l s of different Lewis groups for fucosyltransferase act i v i t i e s demonstrated that GDP-L-fucose: galactosyl-/? - 1 - 4 D-glucose α-3-fucosyltransferase activity is expressed o n l y when t h e i n d i v i d u a l has a L e w i s gene ( 5 1 . ) · ~ p r e s e n c e o f t h e two enzymes meant t h a t i t was n o t p o s s i b l e from these experiments w i t h whole s a l i v a to deduce w h e t h e r the L e w i s enzyme c o u l d t r a n s f e r f u c o s e t o the 0T


n

e

c

o


42

3-, as well as to the 0-4-, position of N-acetyl glucosamine. The c a p a c i t y o f a h i g h l y p u r i f i e d fucosyltransfer a s e f r o m human m i l k t o t r a n s f e r L-fucose to N-acetyl g l u c o s a m i n e i n b o t h T y p e 1 a n d T y p e 2 c h a i n s lecT P r i e e l s e t a l . ( 5 2 ) t o c o n c l u d e t h a t t h e L e - g e n e e n c o d e d enzyme i s a n a-375-fucosyltransferase that can synthesise both X and L e s t r u c t u r e s . In our l a b o r a t o r y , however, further f r a c t i o n a t i o n on S e p h a c r y l S-200 o f a s i m i l a r l y p u r i f i e d preparation of α - 3 / 4 - f u c o s y l t r a n s f e r a s e f r o m human m i l k removed a large proportion of the enzyme activity responsible f o r the transfer o f L-fucose to the O p p o s i t i o n o f the subterminal N-acetylglucosamine residue in l o w m o l e c u l a r w e i g h t Type 2 o l i g o s a c c h a r i d e s ; the r e s u l t a n t enzyme r e a c t e d m a i n l y w i t h N - a c e t y l g l u c o s a m i n e i n Type 1 c h a i n s and D - g l u c o s e i n l a c t o s e - b a s e d struct ures and had no d e t e c t a b l e c a p a c i t y t o t r a n s f e r fucose t o Type 2 chains i n glycoproteins (Table I) (53-54). A s i m i l a r l y p u r i f i e d 3 / 4 - f u c o s y l t r a n s f e r a s e from Âïï"3r~cells b e h a v e d i n t h e same w a y t o w a r d s T y p e 2 s u b s t r a t e s ( T a b l e I) (55). The a c c e p t o r specificity o f the further purified Lewis 3/4-fucosyltransferase therefore suggested that the L e - g e n e e n c o d e d enzyme w o u l d n o t have t h e c a p a c i t y to synthesise X-haptenic structures to any appreciable extent i n vivo. However, Kukowska-Latallo et al.(8) r e c e n t l y c l o n e d the Le gene from the A431 c e l l l i n e u s i n g a mammalian gene t r a n s f e r p r o c e d u r e and d e m o n s t r a t e d that C 0 S - 1 c e l l s t r a n s f e c t e d w i t h t h i s g e n e 1) e x p r e s s both Le and X d e t e r m i n a n t s t h a t were n o t e x p r e s s e d by t h e u n transfected cell a n d 2) c o n t a i n e d a fucosyltransferase t h a t r e a c t e d w i t h b o t h Type 1 and Type 2 s u b s t r a t e s . T h i s a p p a r e n t d i s c r e p a n c y r e m a i n s t o b e r e s o l v e d . One p o s s i b l e e x p l a n a t i o n i s t h a t t h e s o l u b l e enzyme i n m i l k h a s b e e n d i g e s t e d b y p r o t e a s e s t o r e l e a s e i t f r o m t h e G o l g i memb r a n e s a n d t h i s p r o c e s s h a s r e s u l t e d i n some c o n f o r m a t i o n a l change i n t h e enzyme p r o t e i n t h a t i n f l u e n c e s its fine specificity.


a

a

Pathway o f B i o s y n t h e s i s o f S i a l y l - X S t r u c t u r e s . T h e a - 3 / 4 - f u c o s y l t r a n s f e r a s e p r e p a r a t i o n p u r i f i e d f r o m human m i l k by P r i e e l s e t a l . ( 5 2 ) was s a i d n o t t o t r a n s f e r f u c o s e t o s i a l i c a c i d - c o n t a i n i n g s u b s t r a t e s and from t h i s result the pathway of biosynthesis o f s i a l y l - X was u n - c l e a r s i n c e i t h a d p r e v i o u s l y b e e n shown t h a t t h e X (Gal-/3-l-4[Fuc-a-1-3]GlcNAc) structure i s not a substrate f o r the 6 or 3 -sialyltransferases ( 5 6 ) . However, Johnson et al. (57), using a different procedure for assay anct i d e n t i f i c a t i o n o f the product, found that p a r t i a l l y p u r i fied 3/4-fucosyltransferase f r o m human m i l k c o u l d transf e r f u c o s e t o NeuAc a - 2 - 3 G a l - / 3 l - 4 G l c N A c t o f o r m s i a l y l - X , a l t h o u g h N e u A c - a - 2 - 6 G a l - j g - l - 4 G l c N A c was n o t a substrate. The 3 - f u c o s y l t r a n s f e r a s e i n human m i l k , separable from the 3 / 4 - f u c o s y l t r a n s f e r a s e (J58), t h e c o r r e s p o n d i n g e n z f

1



4. WATKINS ET AL.

43


ymes p u r i f i e d f r o m p l a s m a a n d l i v e r ( 5 7 , 5 9 ) ( T a b l e II), t h e e n z y m e i n h u m a n l u n g c a r c i n o m a c e l l s ("27) a n d a n 3 f u c o s y l t r a n s f e r a s e i n h u m a n a m n i o t i c f l u i d Ç5U) w e r e a l s o shown t o u t i l i s e compounds w i t h t e r m i n a l N e u A c - a - 2 - 3 G a l β l-4GlcNAc sequences to form s i a l y l - X structures (Figure 3). More r e c e n t l y M o l l i c o n e et a l . ( 6 1 ) r e - e x a m i n e d homogenates of various tissues for 3-fucosyltransferase a c t i v i t y and c o n f i r m e d the u t i l i s a t i o n o f s i a l y l acc e p t o r s by the α - 3 - e n z y m e s i n plasma and l i v e r and by the 3/4-enzymes i n m i l k , k i d n e y , and g a l l b l a d d e r . The p a t t e r n o f b i o s y n t h e s i s o f s i a l y l - X t h u s r e s e m b l e s the formation o f the d i f u c o s y l L e and Y s t r u c t u r e s i n s o f a r as i n t h e s e d e t e r m i n a n t s t h e a d d i t i o n o f the

Carbohydrate Antigens - American Chemical Society

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