Vol. 85, No. 1, 1978
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
November 14, 1978
Pages 174-182
CONCANAVALIN A MEDIATED UPTAKE OF ENZYMES BY FIBROBLASTS
A l f o n s o G o n z a l e z - N o r i e g a a n d William S. S l y
F r o m the E d w a r d M a l l i n e k r o d t D e p a r t m e n t of P e d i a t r i e s , t h e D e p a r t m e n t of G e n e t i c s , W a s h i n g t o n U n i v e r s i t y School of M e d i c i n e , t h e D i v i s i o n of M e d i c a l G e n e t i c s , St. L o u i s C h i l d r e n ' s H o s p i t a l , St. L o u i s , M i s s o u r i
Received September 23,1978 SUMMARY: C o n e a n a v a l i n A (Con A) e n h a n c e d t h e b i n d i n g a n d u p t a k e of g l y c o p r o t e i n s b y h u m a n f i b r o b l a s t s at d o s e s w h i c h d i d not affect t h e u p t a k e of two n o n - g l y c o s y l a t e d p r o t e i n s . When Con A w a s p r e i n c u b a t e d w i t h g l y c o p r o t e i n s a n d the m i x t u r e l a t e r a d d e d to c u l t u r e d c e l l s , Con A - m e d i a t e d u p t a k e w a s t i m e - , t e m p e r a t u r e - , a n d l e e t i n - e o n e e n t r a t i o n d e p e n d e n t . ~ - M e t h y l m a n n o s i d e (ca'VIM), at c o n c e n t r a t i o n s w h i c h h a v e no effect on t h e p h o s p h o m a n n o s y l - r e e o g n i t i o n s y s t e m for u p t a k e of a c i d h y d r o l a s e s , i n h i b i t e d Con A - m e d i a t e d u p t a k e . T h e s e s t u d i e s i l l u s t r a t e t h a t t h e a n a l y s i s of a d s o r p t i v e p i n o c y t o s i s of g l y c o p r o t e i n s i s o l a t e d on Con A - S e p h a r o s e may b e c o m p l i c a t e d b y Con A - m e d i a t e d u p t a k e of g l y c o p r o t e i n s o t h e r w i s e not taken up. INTRODUCTION:
H i c k m a n a n d N e u f e l d (1) s u g g e s t e d t h a t m a n y l y s o s o m a l h y d r o -
l a s e s h a v e a "common r e c o g n i t i o n m a r k e r " , e s s e n t i a l for t h e i r u p t a k e b y f i b r o b l a s t s . S t u d i e s w i t h h u m a n p l a t e l e t B - g l u c u r o n i d a s e (2) s u g g e s t e d that a d s o r p t i v e p i n o e y t o s i s of t h i s e n z y m e b y f i b r o b l a s t s i n v o l v e s r e c o g n i t i o n of 6 - p h o s p h o m a n n o s e r e s i d u e s on forms of the e n z y m e s u s c e p t i b l e to p i n o c y t o s i s - c a l l e d " h i g h u p t a k e forms" (3).
E x t e n s i o n of t h e s e f i n d i n g s to o t h e r h y d r o l a s e s ( 4 , 5 , 6 ) l e d u s to p o s t u l a t e
t h a t 6 - p h o s p h o m a n n o s e w a s p a r t of t h e common r e c o g n i t i o n m a r k e r for u p t a k e ( 4 ) . O t h e r forms of a c i d h y d r o l a s e s a p p e a r to l a c k t h i s m a r k e r a n d a r e t a k e n up at v e r y low r a t e s b y f i b r o b l a s t s ( 1 , 3 , 4 , 7 ) .
In f a e t , in 8 - g l u e u r o n i d a s e e x t r a c t e d from most
t i s s u e s , most of the c a t a l y t i c a e t i v i t y r e s i d e s in "low u p t a k e forms" w h i c h a r e t a k e n u p v e r y p o o r l y b y f i b r o b l a s t s (3). F i b r o b l a s t s from p a t i e n t s w i t h I - c e l l d i s e a s e s e c r e t e m a n y a c i d h y d r o l a s e s w h i c h are recognition defective (1).
T h i s w a s t h e o b s e r v a t i o n that l e d H i c k m a n a n d
0006-291x/7810851-0174501.OO/O Copyright © 1978 by Academic" Press, Inc. All rights o] reproduction in any Jorm reserved.
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Neufeld to s u g g e s t that m a n y a c i d h y d r o l a s e s s h a r e a common r e c o g n i t i o n m a r k e r for uptake by fibroblasts.
In the c o u r s e of e x p e r i m e n t s c a r r i e d out with I - c e l l s e c r e t i o n
h e x o s a m i n i d a s e i n w h i c h the e n z y m e was i s o l a t e d b y a d s o r p t i o n to, a n d e l u t i o n from C o n e a n a v a l i n A - S e p h a r o s e , we d i s c o v e r e d a n a c t i v i t y w h i c h a p p e a r e d to c o n v e r t I - c e l l h e x o s a m i n i d a s e into a " h i g h - u p t a k e " form for f i b r o b l a s t s .
This activity could
also c o n v e r t h u m a n p l a c e n t a l B - g i u e u r o n i d a s e i n t o " h i g h - u p t a k e " f o r m s .
The initial
a n d i n c o r r e c t i n t e r p r e t a t i o n was that a n e n z y m a t i e a c t i v i t y was i n v o l v e d i n the c o n v e r s i o n p h e n o m e n o n . S u b s e q u e n t e x p e r i m e n t s led u s to i d e n t i f y the a g e n t that s t i m t l l a t e s the u p t a k e of the " l o w - u p t a k e " e n z y m e s as Con A w h i c h eame off the Con A - S e p h a r o s e c o l u m n with the e l u t e d e n z y m e (8).
In this r e p o r t , we p r e s e n t
e x p e r i m e n t s w h i c h d e m o n s t r a t e Con A m e d i a t e d u p t a k e of ~ - g l u c u r o n i d a s e , I - c e l l h e x o s a m i n i d a s e , m a n n o s e - a l b u m i n g l y c o c o n j u g a t e (Man-BSA) a n d r i b o n u c l e a s e B (RNase B ) . MATERIALS AND METHODS: Human p l a c e n t a B - g l u c u r o n i d a s e was p r e p a r e d as p r e v i o u s l y d e s c r i b e d (4). N - a c e t y l - ~ - D - h e x o s a m i n i d a s e ( d e s i g n a t e d b e l o w as h e x o s a m i n i d a s e ) from I - c e l l f i b r o b l a s t s e c r e t i o n s was collected as d e s c r i b e d b y K a p l a n et al. (4), c o n c e n t r a t e d b y u l t r a f i l t r a t i o n a n d a p p l i e d to a c o l u m n of c o n e a n a v a l i n A - S e p h a r o s e ( P h a r m a c i a ) . After r e m o v a l of u n a d s o r b e d p r o t e i n , h e x o s a m i n i d a s e a c t i v i t y was e l u t e d with O. 75 M ~ - m e t h y l - D - m a n n o s i d e i n 0.15 M NaCI, 10 mM NazHPO 4, 20 mM T r i s - H C l b u f f e r , pH 7 . 5 . H e x o s a m i n i d a s e - f r e e h u m a n s e r u m a l b u m i n (The A m e r i c a n National Red C r o s s ) was a d d e d to the f i b r o b l a s t s e c r e t i o n m e d i u m (final c o n c e n t r a t i o n 1 m g / m l ) p r i o r to u l t r a f i l t r a t i o n . R i b o n u c l e a s e B (Sigma t y p e XII-B) was r e p u r i f i e d on e o n c a n a v a l i n A - S e p h a r o s e to r e m o v e a n y r i b o n u e l e a s e A (RNase A) w h i c h was s h o w n to b e p r e s e n t (9). To e l i m i n a t e p o s s i b l e Con A c o n t a m i n a t i o n both RNase A a n d RNase B w e r e s u b j e c t e d to u l t r a f i l t r a t i o n on XM-50 m e m b r a n e f i l t e r s ( A m i e o n ) , e o n e e n t r a t e d on UM-2 m e m b r a n e f i l t e r s ( A m i e o n ) , c h r o m a t o g r a p h e d on S e p h a d e x G-75 (1.5x58 cm) followed b y u l t r a f i l t r a t i o n a n d e x h a u s tive d i a l y s i s a g a i n s t 20 mM Na2HPO 4, pH 7 . 6 . M a n n o s e - B S A (gift of D r . Y. C. Lee) (10), b o v i n e s e r u m a l b u m i n , R N a s e A a n d RNase B w e r e i o d i n a t e d u s i n g the c h l o r o m i n e T method (11). Speeifie a c t i v i t i e s w e r e 6 _+.3 u C i / ~ g for RNase A a n d B a n d 36+6 ~ Ci/~ g for BSA a n d Man-BSA. B - G l u e u r o n i d a s e a n d B - h e x o s a m i n i d a s e d e f i c i e n t h u m a n f i b r o b l a s t s w e r e g r o w n as d e s c r i b e d p r e v i o u s l y (2). P i n o c y t o s i s m e a s u r e m e n t s : The i n d i c a t e d p r o t e i n s w e r e i n c u b a t e d w i t h Con A (Sigma, g r a d e IV) 20 rain at room t e m p e r a t u r e i n the p r e s e n c e of 250 ~ g h u m a n s e r u m a l b u m i n , 0.15 M NaCl, 10 mM NazHPO 4, I0 mM T r i s - H C 1 , pH 7 . 6 , i n a f i n a l v o l u m e of 0.2 ml. One ml of E a g l e ' s Minimal E s s e n t i a l Medium c o n t a i n i n g 0.2% h u m a n s e r u m a l b u m i n was t h e n a d d e d a n d 1 ml of t h i s m i x t u r e was l a y e r e d onto c u l t u r e d f i b r o b l a s t s i n 35 mm p e t r i d i s h e s . The c e l l s w e r e a p p r o x i m a t e l y 75% c o n f l u e n t . After 1 h r i n c u b a t i o n at 37°C, d i s h e s w e r e c h i l l e d a n d w a s h e d s i x times w i t h cold p h o s p h a t e - b u f f e r e d s a l i n e . Cell a s s o c i a t e d a c t i v i t y was m e a s u r e d as d e s c r i b e d b y K a p l a n et a l . (2). ~ - G l u c u r o n i d a s e a n d h e x o s a m i n i d a s e w e r e a s s a y e d as d e s c r i b e d b y G l a s e r et al. (12) a n d p r o t e i n a c c o r d i n g to L o w r y et al. (13). R a d i o a c t i v i t y was m e a s u r e d i n a P a c k a r d gamma c o u n t e r . T h e r a t e of b i n d i n g a n d / o r u p t a k e is e x p r e s s e d as the p e r c e n t of a d d e d r a d i o a c t i v i t y or e n z y m a t i c a c t i v i t y w h i c h was cell a s s o c i a t e d p e r u n i t time (% u p t a k e / r a g protein/hr).
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60 CON Aj.o
_z I.d k0 40 ¢D
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F i g u r e 1. Cells w e r e w a s h e d with cold T r i s - p h o s p h a t e b u f f e r e d s a l i n e (TPBS) a n d t h e n i n c u b a t e d with 0.5 ml of TPBS c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s of ConAwith (•) or w i t h o u t ( O ) 50ram aMM. After 15 m i n a t 4°C d i s h e s w e r e w a s h e d a n d i n c u b a t e d with 1700 U of B - g l u e u r o n i d a s e in 0.5 ml of TPBS. At the e n d of the s e c o n d m e u b a t l o n perxod (15 mln at 4 C ) , p l a t e s w e r e w a s h e d a n d cell a s s o c i a t e d a c t i v i t y was m e a s u r e d .
RESULTS AND DISCUSSION: B i n d i n g of p l a c e n t a l ~ - g l u e u r o n i d a s e to Con A p r e e o a t e d cells - H u m a n p l a c e n t a l B - g l u e u r o n i d a s e w h i c h is p r e d o m i n a n t l y a " l o w - u p t a k e " form for f i b r o b l a s t s does nol b i n d d e t e e t a b l y to the cell s u r f a c e of h u m a n f i b r o b l a s t s . F i g u r e 1 s h o w s that p r i o r t r e a t m e n t of h u m a n f i b r o b l a s t s with Con A at 4°C a n d s u b s e q u e n t r e m o v a l of u n b o u n d l e e t i n b y e x t e n s i v e w a s h i n g , l e a d s to d r a m a t i c b i n d i n g of e n z y m e a d d e d to the f i b r o b l a s t s i n a s e c o n d i n c u b a t i o n at 4°C.
The a m o u n t of e n z y m e b o u n d is p r o p o r -
t i o n a l to the Con A c o n c e n t r a t i o n u s e d i n the p r i o r i n c u b a t i o n . E s s e n t i a l l y no e n zyme b i n d i n g o c c u r r e d w h e n 50 mM c~IM h a d b e e n p r e s e n t d u r i n g the e x p o s u r e of f i b r o b l a s t s to Con A.
T h e s e data s u g g e s t that the Con A s e r v e s as a b r i d g e b e t w e e n
cell s u r f a c e g l y e o p r o t e i n s a n d the " l o w - u p t a k e " ~ - g l u e u r o n i d a s e . Con A m e d i a t e d u p t a k e of " l o w - u p t a k e " B - g l u e u r o n i d a s e - It h a s b e e n p r e v i o u s l y e s t a b l i s h e d that Con A c a n b i n d to the s u r f a c e m e m b r a n e (14,15) a n d b e i n t e r n a l ized (16) b y f i b r o b l a s t s i n e u l t u r e .
H u m a n p l a c e n t a l B - g l u e u r o n i d a s e is i t s e l f ,
v e r y p o o r l y t a k e n u p b y h u m a n f i b r o b l a s t s (0.2% U / r a g / h ) .
H o w e v e r , m i x i n g the
e n z y m e w i t h free Con A i n a p r i o r i n c u b a t i o n a n d s u b s e q u e n t e x p o s u r e of cells to
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I00~ C ~
8O
.®
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F i g u r e 2. A. Con A m e d i a t e d b i n d i n g (4°C) a n d u p t a k e (37°C) of ~ - g l u c u r o n i d a s e . Cells i n 35 mm d i s h e s w e r e i n e u b a t e d for l h r at 4°C ( • ) a n d 37°C ( • ) with 1 ml of TPBS c o n t a i n i n g ~ - g l u c u r o n i d a s e (1700 U ) , Con A at the c o n c e n t r a t i o n s i n d i c a t e d a n d h u m a n s e r u m a l b u m i n (25 ~ g ) . T h e ~ - g l u e u r o n i d a s e a n d Con A h a d b e e n p r e i n e u b a t e d i n 0.2 ml of TPBS for 20 m i n at room t e m p e r a t u r e b e f o r e d i l u t i o n a n d a d d i n g to c e l l s . B. Time d e p e n d e n c e of Con A mediated e n z y m e u p t a k e . Con A (5 ~ g ) , p r e i n e u b a t e d with 1600 U of 8 - g l u c u r o n i d a s e was a d d e d to cells i n the p r e s e n c e of ( • ) or i n the a b s e n c e of ( • , • ) 50 mM c~IM. At the e n d of the i n c u b a t i o n p e r i o d cells w e r e w a s h e d with b u f f e r e d s a l i n e with ( • , • ) or w i t h o u t ( • ) 50 mM ~/M. C. I n h i b i t i o n of the Con A m e d i a t e d u p t a k e b y s u g a r s . Con A (5 g g) w a s p r e i n e u b a t e d with 1600 U of B - g l u c u r o n i d a s e a n d t h e n e x p o s e d to cells i n the p r e s e n c e of m a n n o s e ( • ) , m a n n o s e - 6 - p h o s p h a t e ( • ) or c~VIM ( • ) . Cell a s s o c i a t e d B - g l u e u r o n i d a s e was m e a s u r e d after a 1 h o u r i n c u b a t i o n at 37°C.
the e n z y m e - l e e t i n m i x t u r e p r o d u e e d an i n c r e a s e i n c e l l - a s s o c i a t e d e n z y m e .
Figure
2-A shows the effect of i n c r e a s i n g c o n c e n t r a t i o n s of Con A on the a m o u n t of c e l l - a s s o ciated e n z y m e w h e n the c e l l s w e r e e x p o s e d to the m i x t u r e s at 4°C a n d 37°C. At 4°C, w h e r e c e l l - a s s o c i a t e d e n z y m e p r e s u m a b l y r e p r e s e n t s e n z y m e b o u n d b u t not i n t e r n a l i z e d , maximum s t i m u l a t i o n of b i n d i n g o e e u r r e d at 5-8 ~ g / m l Con A.
Washing
the d i s h e s with PBS c o n t a i n i n g 50 mM ~MM after the i h o u r i n c u b a t i o n p e r i o d at 4°C, r e l e a s e d 29% of the c e l l - a s s o c i a t e d e n z y m e b o u n d with 5 g g / m l of Con A.
At 37°C
the l e c t i n p r o d u c e d a much l a r g e r i n c r e a s e i n c e l l - a s s o e i a t e d e n z y m e . A g a i n , the maximum s t i m u l a t i o n o e e u r r e d b e t w e e n 5-8 ~ g / m l ; b u t h i g h e r l e v e l s led to r e d u c e d uptake.
In t h i s e x p e r i m e n t , e n z y m e w h i c h b e c a m e c e l l - a s s o c i a t e d d u r i n g the 1 h o u r
i n c u b a t i o n at 37°C p r e s u m a b l y r e f l e c t e d the sum of e n z y m e b o u n d to the cell s u r face as a e o n s e q u e n c e of the i n t e r a c t i o n with the l e e t i n , a n d e n z y m e i n t e r n a l i z e d a f t e r
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binding.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In a g r e e m e n t with the e x p e c t a t i o n from this i n t e r p r e t a t i o n , o n l y 6% of the
e n z y m e t a k e n u p after 1 h o u r i n the p r e s e n c e of 5 la g / m l Con A was r e l e a s a b l e b y w a s h i n g with 50 mM aMM. Time d e p e n d e n c e of Con A m e d i a t e d ~ - g l u c u r o n i d a s e u p t a k e - F i g u r e 2-B s h o w s the time d e p e n d e n e e of Con A m e d i a t e d u p t a k e of p l a c e n t a l B - g l u c u r o n i d a s e at 37°C. T h e e n z y m e t a k e n u p , w h i c h i n c l u d e s the sum of b o u n d a n d i n t e r n a l i z e d e n z y m e , i n c r e a s e d l i n e a r l y u n d e r t h e s e c o n d i t i o n s for at least 60 rain. A d d i t i o n of aMM (50 mM), at the time the p r e s u m p t i v e e n z y m e - Con A complex was a d d e d to p l a t e s , l a r g e l y p r e v e n t e d the time d e p e n d e n t u p t a k e .
In c o n t r a s t , a d d i t i o n of the s u g a r
after the i n c u b a t i o n p e r i o d r e l e a s e d o n l y a small f r a c t i o n (6-17%) of the f i b r o b l a s t a s s o c i a t e d e n z y m e . T h i s o b s e r v a t i o n s u g g e s t e d that most of the cell a s s o c i a t e d e n zyme had b e e n i n t e r n a l i z e d d u r i n g the i n c u b a t i o n at 37°C. I n h i b i t i o n of the Con A m e d i a t e d u p t a k e b y s i m p l e s u g a r s - F i g u r e 2-C c o m p a r e s the i n h i b i t i o n of p l a c e n t a l ~ - g l u c u r o n i d a s e u p t a k e b y s u g a r s p r e s e n t i n the m e d i u m to w h i c h the Con A / B - g l u c u r o n i d a s e m i x t u r e was a d d e d ,
aMM was c l e a r l y the most
p o t e n t i n h i b i t o r , about 5 fold more p o t e n t t h a n the o t h e r s u g a r s b a s e d on the concent r a t i o n p r o d u c i n g half of maximum i n h i b i t i o n . T h e s e r e s u l t s c o n t r a s t s h a r p l y with the r e s u l t s of t h e s e i n h i b i t o r s on the a d s o r p t i v e p i n o e y t o s i s of acid h y d r o l a s e s b y the p h o s p h o m a n n o s y l r e c o g n i t i o n s y s t e m i n f i b r o b l a s t s . T h e r e , m a n n o s e ~ 6 - P i s about 1,000 times more potent t h a n m a n n o s e or aMM as an i n h i b i t o r of e n z y m e p i n o c y t o s i s (2). Effect of Con A on the u p t a k e of o t h e r p r o t e i n s - E d e l s o n a n d Cohn (17) r e p o r t e d that Con A p r o d u c e d a four fold e n h a n c e m e n t of u p t a k e of h o r s e r a d i s h p e r o x i d a s e (HRP) b y mouse p e r i t o n e a l m a e r o p h a g e s .
T h e y did not a t t r i b u t e this s t i m u l a t i o n to
the i n t e r a c t i o n b e t w e e n Con A a n d the m a n n o s e - r i c h g l y e o p r o t e i n (HRP), s i n c e they o b s e r v e d s i m i l a r s t i m u l a t i o n of [1251] BSA u p t a k e . We h a v e e x a m i n e d the effects of Con A on two p r o t e i n s w h i c h c o n t a i n no c a r b o h y d r a t e , a n d also on s e v e r a l g l y e o p r o t e i n s . In F i g . 3 we c o m p a r e the s t i m u l a t i o n of u p t a k e b y Con A for [125I] - m a n n o s e - B S A , a n d [1251]RNaseB.
RNaseBhas
a s i n g l e o l i g o s a e c h a r i d e c h a i n a n d is q u a n t i t a t i v e l y r e t a i n e d on Con A - S e p h a r o s e
178
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I,G o~ -r ¢lJ
0.8 ~
co.= O E
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I0
20 CON
30 A (/~G/ML)
9
40
'$~ 50
F i g u r e 3. Effect of Con A on u p t a k e of g l y c o s y l a t e d a n d n o n - g l y c o s y l a t e d p r o t e i n s . E x p e r i m e n t s w e r e p e r f o r m e d a s d e s c r i b e d in F i g . 2, Con A w a s p r e i n c u b a t e d in the p r e s e n c e of 5 p m o l e s o f [ 1 2 5 I ] B S A ( A ) , [ 1 2 5 1 ] M a n - B S A (•), 125I]RNaseA ( O ) o r [ I25 I]RNaseB ( • ), before the mlxturewas added to t h e d i s h e s . Cell a s s o c i a t e d r a d i o a c t i v i t y m e a s u r e d a f t e r a 1 h o u r i n c u b a t i o n at 37°C. columns.
r
3
O n l y 16% of the L125ij Man-BSA c o n t a i n i n g an a v e r a g e of 20 m a n n o s e s u b -
s t i t u t i o n s was r e t a i n e d b y Con A - S e p h a r o s e a n d s p e c i f i c a l l y e l u t e d .
S t i m u l a t i o n of
u p t a k e b y Con A i s s e e n b o t h w i t h [1251] RNase B a n d [1251] M a n - B S A , b u t not w i t h t h e c o r r e s p o n d i n g n o n c a r b o h y d r a t e c o n t a i n i n g p r o t e i n s ( F i g . 3).
Note t h a t t h e
s t i m u l a t i o n s e e n w i t h t h e s e g l y e o p r o t e i n s is v e r y s m a l l c o m p a r e d to t h a t for ~ - g l u c u r o n i d a s e ( F i g . 2) o r I - c e l l s e c r e t i o n h e x o s a m i n i d a s e ( F i g . 4 ) .
Figure 4 (A-C)
p r e s e n t s e x p e r i m e n t s s h o w i n g the effect of Con A on t h e u p t a k e of t h r e e d i f f e r e n t g l y c o p r o t e i n s , RNase B, h u m a n p l a c e n t a l ~ - g l u c u r o n i d a s e , a n d h e x o s a m i n i d a s e from I - c e l l s e c r e t i o n s . w e l l c h a r a c t e r i z e d (18).
T h e s i n g l e o l i g o s a c c h a r i d e c h a i n of RNase B h a s b e e n Human p l a c e n t a l ~ - g l u c u r o n i d a s e i s a t e t r a m e r w h i c h c o n -
t a i n s 6.5% c a r b o h y d r a t e s , a n d is m a n n o s e - r i e h (19). s a c c h a r i d e c h a i n s p e r m o n o m e r i s not k n o w n .
H o w e v e r , the n u m b e r of oligo-
Little o r n o t h i n g i s k n o w n of t h e
c a r b o h y d r a t e c h a i n c o m p o s i t i o n of I - c e l l s e c r e t i o n h e x o s a m i n i d a s e .
H o w e v e r , it i s
q u a n t i t a t i v e l y a d s o r b e d from c u l t u r e m e d i u m b y Con A - S e p h a r o s e (A. G o n z a l e z N o r i e g a a n d W. S. S l y , u n p u b l i s h e d o b s e r v a t i o n s ) .
The stimulation, by increasing
c o n c e n t r a t i o n s of Con A , of u p t a k e of t h e s e t h r e e d i f f e r e n t g l y c o p r o t e i n s , s t u d i e d e i t h e r i n d i v i d u a l l y o r in t h e p r e s e n c e of a s e c o n d g l y c o p r o t e i n , i s s h o w n in F i g . 4. T h e p a t t e r n of s t i m u l a t i o n a p p e a r e d d i s t i n c t i v e for e a c h of t h e t h r e e g l y c o p r o t e i n s studied.
A d d i t i o n of a s e c o n d g l y c o p r o t e i n i n f l u e n c e d the l e v e l of s t i m u l a t i o n s e e n
179
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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
14
~ RNase B ALONE
/
LOCU"ON'OASE i I"I0
rr
I0 Z
-®
W I-0
HEXOSAMINIDASE ALONE -m- + ,8-GLUCURONIDASE + RNase B
L9 W < I-n
I
I
28 4 - /9-GLUCURONIDASE ALONE --o- + HEXOSAMINIDASE se B
20
12 e
5'o
•
,~o
,~o
26o
a~o
CON A (p.G/ML)
F i g u r e 4. P e r c e n t of cell a s s o c i a t e d [ 1251] RNase B (A), h e x o s a m i n i d a s e (B) a n d ~ - g l u c u r o n i d a s e (C) w h e n the i n d i c a t e d c o n c e n t r a t i o n of Con A was p r e i n c u b a t e d with each g l y c o p r o t e i n s i n g l y or with one of the o t h e r two g l y c o p r o t e i n s . Amount of each a d d e d w h e n p r e s e n t : [1251] RNase B (5 p m o l e s ) , h e x o s a m i n i d a s e (1300 U) B - g l u c u r o n i d a s e (1600 U ) . Cell a s s o c i a t e d e n z y m e a c t i v i t y or r a d i o a c t l v l t y w e r e m e a s u r e d after a 1 h o u r x n c u b a t l o n at 37 C.
(in all 3 c a s e s e n h a n c i n g u p t a k e ) , b u t did not a p p e a r to a l t e r the p a t t e r n of u p t a k e of the f i r s t l i g a n d .
We h a v e no e x p l a n a t i o n for t h i s effect.
Con A is k n o w n to b e a t e t r a v a l e n t l e c t i n (20).
As s u c h , it might b e e x p e c t e d
to s e r v e as a c r o s s - l i n k i n g a g e n t b e t w e e n cell s u r f a c e g l y c o p r o t e i n s a n d some glyco p r o t e i n s i n the m e d i u m .
S u c h r e a c t i o n s c o u l d b e q u i t e complicated s i n c e e i t h e r the
cell s u r f a c e g l y c o p r o t e i n or the p r o t e i n i n the medium c a n b e m o n o v a l e n t or m u l t i -
180
Vol. 85, No. 1, 1978
valent itself.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
P r e s u m a b l y s u c h d i f f e r e n c e s i n the e n z y m e s e x p l a i n s the d i f f e r e n t
p a t t e r n s of s t i m u l a t i o n b y Con A. Beeck et a l . (21) r e p o r t e d an a b s t r a c t on the effects of Con A a n d s u e c i n y l Con A on e n d o c y t o s i s of l y s o s o m a l e n z y m e s . T h e i r s t u d i e s d i f f e r e d from those p r e s e n t e d here.
The e n z y m e s u s e d w e r e s u b j e c t to a d s o r p t i v e e n d o c y t o s i s i n the a b s e n c e of
l e c t i n ( i . e . w e r e " h i g h u p t a k e " forms) a n d t h e y d e s c r i b e d i n h i b i t i o n of e n z y m e p i n o c y t o s i s b y the l e c t i n s .
T h e s e effects w e r e a t t r i b u t e d to i n t e r a c t i o n s of the l e c t i n with
both the r e c o g n i t i o n m a r k e r on the e n z y m e s a n d the f i b r o b l a s t r e c e p t o r .
We i n t e n -
t i o n a l l y r e s t r i c t e d o u r s t u d i e s to the effects of Con A on p i n o c y t o s i s of g l y c o p r o t e i n s w h i c h are not s u b j e c t to a d s o r p t i v e e n d o c y t o s i s . O u r s t u d i e s d e m o n s t r a t e d d r a m a t i c s t i m u l a t i o n of p i n o c y t o s i s of t h e s e g l y c o p r o t e i n s b y Con A u n d e r c o n d i t i o n s w h e r e u p t a k e of n o n - g l y c o p r o t e i n s was not affected.
We i n t e r p r e t t h e s e o b s e r v a t i o n s to
i n d i c a t e that the l e c t i n can s e r v e as a b r i d g e b e t w e e n a l e e t i n b i n d i n g site on the cell s u r f a c e a n d a l e c t i n b i n d i n g site on the g l y c o p r o t e i n a n d lead to u p t a k e of the glycoprotein by "piggyback endoeytosis." The f i n d i n g that Con A c a n mediate u p t a k e of a g l y c o p r o t e i n o t h e r w i s e not s u s c e p t i b l e to a d s o r p t i v e p i n o e y t o s i s has i m p o r t a n t p r a c t i c a l i m p l i c a t i o n s . Many g l y c o p r o t e i n s a r e i s o l a t e d b y affinity c h r o m a t o g r a p h y u s i n g Con A - S e p h a r o s e .
S i n c e free
Con A is k n o w n to come off s u c h c o l u m n s (8), s t u d i e s of e n z y m e u p t a k e u s i n g e n zymes i s o l a t e d on Con A - S e p h a r o s e c o l u m n s must be i n t e r p r e t e d v e r y c a u t i o u s l y . One must p r o v i d e e v i d e n c e that no free Con A is p r e s e n t i n the e n z y m e p r e p a r a t i o n , or that the u p t a k e is not i n h i b i t e d b y ~MM. A p r a c t i c a l w a y to d i s t i n g u i s h Con A m e d i a t e d u p t a k e b y f i b r o b l a s t s is to i n c l u d e 25 mM ~//M i n the i n c u b a t i o n . T h a t c o n c e n t r a t i o n is too low to i n h i b i t the u p t a k e b y the 6 - p h o s p h o m a n n o s y l r e c o g n i t i o n s y s t e m (2) b u t effectively b l o c k s the Con A m e d i a t e d u p t a k e w h i c h we h a v e d e s c r i b e d here.
ACKNOWLEDGEMENTS: T h i s w o r k is s u p p o r t e d i n p a r t b y the U . S . P . H . S . g r a n t 21096 a n d the R a n k e n J o r d a n F u n d for the C r i p p l i n g D i s e a s e s of C h i l d r e n . T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e the a d v i c e a n d a s s i s t a n c e of D r . D a n i e l A e h o r d , D a v i d F i s c h e r , a n d M a r v i n Natowicz on s e v e r a l a s p e c t s of t h i s w o r k , a n d D r s . R o s a l i n d K o r n f e l d , Paul S c h l e s i n g e r , a n d P h i l i p Stahl for c r i t i c i s m s of the manuscript.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. i0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
H i c k m a n , S. a n d N e u f e l d , E. F . (1972) B i o c h e m . B i o p h y s . R e s . Commun. 49, 992-999. K a p l a n , A . , A c h o r d , D. T . , S l y , W . S . (1977) P r o c . N a t l . A c a d . S c i . USA. 74, 2026-2030. G l a s e r , J . H . , R o o z e n , K. J . , B r o t , F . E . , a n d S l y , W. S. (1975) A r c h . Biochem. B i o p h y s . 166, 536-542. K a p l a n , A . , F i s c h e r , D . , A c h o r d , D. T . , a n d S l y , W. S. (1977) J . C l i n . I n v e s t . 60, 1088-1093. S a n d o , E. N . , N e u f e l d , E. F . (1977) Cell 619-627. U l l r i c h , K . , M e r s m a n n , B . , W e b e r , E . , v o n F i g u r a , K, (1978) B i o c h e m . J . 170, 643-650. N e u f e l d , E. F . , S a n d o , G. N . , G a r v i n , A . J . , a n d Rome, L. H. (1977) J . S t r u c t S u p r a m o l . 6, 95-101. F u r b i s h , F . S . , B l a i r , H. E . , S h i l o a c h , J . , P e n t c h e v , P. G . , a n d B r a d y , R. O. (1977) P r o c . Natl. A c a d . S c i . USA. 74, 3560-3563. B a y n e s , J . W . , a n d Wold, F . (1976) J . B i o l . Chem. 251, 6016-6024. K r a n t z , M. J . , H o l t z m a n , N. A . , S t o w e l l , C. P . , a n d L e e , Y. C. (1976) B i o c h e m i s t r y . 15, 3963-3968. H u n t e r , W. M. a n d G r e e n w o o d , F . C. (1962) N a t u r e 194, 495-496. G l a s e r , J . H . , a n d S l y , W. S. (1973) J . L a b . C l i n . M e d . 82, 969-977. L o w r y , O. H . , R o s e b r o u g h , N. J . , F a r r , A . L . , a n d R a n d a l l , R. J . (1951) J . Biol. Chem. 193, 265-275. I n b a r , M . , a n d S a c h s , L. (1969) P r o c . N a t l . A e a d . S c i . USA. 63, 1418-1425. Noonan, K. D . , a n d B u r g e r , M. M. (1973) J . Biol. Chem. 248, 4286-4292. S t o r r i e , B . , and E d e l s o n , P. J . (1977) Cell 11, 707-717. E d e l s o n , P. J . , a n d C o h n , Z. A . (1974) J . E x p t l . Med. 140, 1364-1368. T a r e n t i n o , A . , P l u m m e r , T . H . , J r . , M a l e y , F . (1970) J . Biol. Chem. 245, 4150-4157. B r o t , F . E . , B e l l , E. C . , J r . , a n d S l y , W. S. (1978) B i o c h e m i s t r y 17, 385-391. A g r a w a l , B. B. L . , a n d G o l d s t e i n , I. J . (1968) A r c h . B i o c h e m . B i o p h y s . 124, 218-219. B e e c k , H . , U l l r i c h , K . , a n d v o n F i g u r a , K. (1977) S y m p o s i u m on B i o l o g y of C o n n e c t i v e T i s s u e , U p p s a l a , S w e d e n , p . 121.
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November 14, 1978
Pages 174-182
CONCANAVALIN A MEDIATED UPTAKE OF ENZYMES BY FIBROBLASTS
A l f o n s o G o n z a l e z - N o r i e g a a n d William S. S l y
F r o m the E d w a r d M a l l i n e k r o d t D e p a r t m e n t of P e d i a t r i e s , t h e D e p a r t m e n t of G e n e t i c s , W a s h i n g t o n U n i v e r s i t y School of M e d i c i n e , t h e D i v i s i o n of M e d i c a l G e n e t i c s , St. L o u i s C h i l d r e n ' s H o s p i t a l , St. L o u i s , M i s s o u r i
Received September 23,1978 SUMMARY: C o n e a n a v a l i n A (Con A) e n h a n c e d t h e b i n d i n g a n d u p t a k e of g l y c o p r o t e i n s b y h u m a n f i b r o b l a s t s at d o s e s w h i c h d i d not affect t h e u p t a k e of two n o n - g l y c o s y l a t e d p r o t e i n s . When Con A w a s p r e i n c u b a t e d w i t h g l y c o p r o t e i n s a n d the m i x t u r e l a t e r a d d e d to c u l t u r e d c e l l s , Con A - m e d i a t e d u p t a k e w a s t i m e - , t e m p e r a t u r e - , a n d l e e t i n - e o n e e n t r a t i o n d e p e n d e n t . ~ - M e t h y l m a n n o s i d e (ca'VIM), at c o n c e n t r a t i o n s w h i c h h a v e no effect on t h e p h o s p h o m a n n o s y l - r e e o g n i t i o n s y s t e m for u p t a k e of a c i d h y d r o l a s e s , i n h i b i t e d Con A - m e d i a t e d u p t a k e . T h e s e s t u d i e s i l l u s t r a t e t h a t t h e a n a l y s i s of a d s o r p t i v e p i n o c y t o s i s of g l y c o p r o t e i n s i s o l a t e d on Con A - S e p h a r o s e may b e c o m p l i c a t e d b y Con A - m e d i a t e d u p t a k e of g l y c o p r o t e i n s o t h e r w i s e not taken up. INTRODUCTION:
H i c k m a n a n d N e u f e l d (1) s u g g e s t e d t h a t m a n y l y s o s o m a l h y d r o -
l a s e s h a v e a "common r e c o g n i t i o n m a r k e r " , e s s e n t i a l for t h e i r u p t a k e b y f i b r o b l a s t s . S t u d i e s w i t h h u m a n p l a t e l e t B - g l u c u r o n i d a s e (2) s u g g e s t e d that a d s o r p t i v e p i n o e y t o s i s of t h i s e n z y m e b y f i b r o b l a s t s i n v o l v e s r e c o g n i t i o n of 6 - p h o s p h o m a n n o s e r e s i d u e s on forms of the e n z y m e s u s c e p t i b l e to p i n o c y t o s i s - c a l l e d " h i g h u p t a k e forms" (3).
E x t e n s i o n of t h e s e f i n d i n g s to o t h e r h y d r o l a s e s ( 4 , 5 , 6 ) l e d u s to p o s t u l a t e
t h a t 6 - p h o s p h o m a n n o s e w a s p a r t of t h e common r e c o g n i t i o n m a r k e r for u p t a k e ( 4 ) . O t h e r forms of a c i d h y d r o l a s e s a p p e a r to l a c k t h i s m a r k e r a n d a r e t a k e n up at v e r y low r a t e s b y f i b r o b l a s t s ( 1 , 3 , 4 , 7 ) .
In f a e t , in 8 - g l u e u r o n i d a s e e x t r a c t e d from most
t i s s u e s , most of the c a t a l y t i c a e t i v i t y r e s i d e s in "low u p t a k e forms" w h i c h a r e t a k e n u p v e r y p o o r l y b y f i b r o b l a s t s (3). F i b r o b l a s t s from p a t i e n t s w i t h I - c e l l d i s e a s e s e c r e t e m a n y a c i d h y d r o l a s e s w h i c h are recognition defective (1).
T h i s w a s t h e o b s e r v a t i o n that l e d H i c k m a n a n d
0006-291x/7810851-0174501.OO/O Copyright © 1978 by Academic" Press, Inc. All rights o] reproduction in any Jorm reserved.
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Neufeld to s u g g e s t that m a n y a c i d h y d r o l a s e s s h a r e a common r e c o g n i t i o n m a r k e r for uptake by fibroblasts.
In the c o u r s e of e x p e r i m e n t s c a r r i e d out with I - c e l l s e c r e t i o n
h e x o s a m i n i d a s e i n w h i c h the e n z y m e was i s o l a t e d b y a d s o r p t i o n to, a n d e l u t i o n from C o n e a n a v a l i n A - S e p h a r o s e , we d i s c o v e r e d a n a c t i v i t y w h i c h a p p e a r e d to c o n v e r t I - c e l l h e x o s a m i n i d a s e into a " h i g h - u p t a k e " form for f i b r o b l a s t s .
This activity could
also c o n v e r t h u m a n p l a c e n t a l B - g i u e u r o n i d a s e i n t o " h i g h - u p t a k e " f o r m s .
The initial
a n d i n c o r r e c t i n t e r p r e t a t i o n was that a n e n z y m a t i e a c t i v i t y was i n v o l v e d i n the c o n v e r s i o n p h e n o m e n o n . S u b s e q u e n t e x p e r i m e n t s led u s to i d e n t i f y the a g e n t that s t i m t l l a t e s the u p t a k e of the " l o w - u p t a k e " e n z y m e s as Con A w h i c h eame off the Con A - S e p h a r o s e c o l u m n with the e l u t e d e n z y m e (8).
In this r e p o r t , we p r e s e n t
e x p e r i m e n t s w h i c h d e m o n s t r a t e Con A m e d i a t e d u p t a k e of ~ - g l u c u r o n i d a s e , I - c e l l h e x o s a m i n i d a s e , m a n n o s e - a l b u m i n g l y c o c o n j u g a t e (Man-BSA) a n d r i b o n u c l e a s e B (RNase B ) . MATERIALS AND METHODS: Human p l a c e n t a B - g l u c u r o n i d a s e was p r e p a r e d as p r e v i o u s l y d e s c r i b e d (4). N - a c e t y l - ~ - D - h e x o s a m i n i d a s e ( d e s i g n a t e d b e l o w as h e x o s a m i n i d a s e ) from I - c e l l f i b r o b l a s t s e c r e t i o n s was collected as d e s c r i b e d b y K a p l a n et al. (4), c o n c e n t r a t e d b y u l t r a f i l t r a t i o n a n d a p p l i e d to a c o l u m n of c o n e a n a v a l i n A - S e p h a r o s e ( P h a r m a c i a ) . After r e m o v a l of u n a d s o r b e d p r o t e i n , h e x o s a m i n i d a s e a c t i v i t y was e l u t e d with O. 75 M ~ - m e t h y l - D - m a n n o s i d e i n 0.15 M NaCI, 10 mM NazHPO 4, 20 mM T r i s - H C l b u f f e r , pH 7 . 5 . H e x o s a m i n i d a s e - f r e e h u m a n s e r u m a l b u m i n (The A m e r i c a n National Red C r o s s ) was a d d e d to the f i b r o b l a s t s e c r e t i o n m e d i u m (final c o n c e n t r a t i o n 1 m g / m l ) p r i o r to u l t r a f i l t r a t i o n . R i b o n u c l e a s e B (Sigma t y p e XII-B) was r e p u r i f i e d on e o n c a n a v a l i n A - S e p h a r o s e to r e m o v e a n y r i b o n u e l e a s e A (RNase A) w h i c h was s h o w n to b e p r e s e n t (9). To e l i m i n a t e p o s s i b l e Con A c o n t a m i n a t i o n both RNase A a n d RNase B w e r e s u b j e c t e d to u l t r a f i l t r a t i o n on XM-50 m e m b r a n e f i l t e r s ( A m i e o n ) , e o n e e n t r a t e d on UM-2 m e m b r a n e f i l t e r s ( A m i e o n ) , c h r o m a t o g r a p h e d on S e p h a d e x G-75 (1.5x58 cm) followed b y u l t r a f i l t r a t i o n a n d e x h a u s tive d i a l y s i s a g a i n s t 20 mM Na2HPO 4, pH 7 . 6 . M a n n o s e - B S A (gift of D r . Y. C. Lee) (10), b o v i n e s e r u m a l b u m i n , R N a s e A a n d RNase B w e r e i o d i n a t e d u s i n g the c h l o r o m i n e T method (11). Speeifie a c t i v i t i e s w e r e 6 _+.3 u C i / ~ g for RNase A a n d B a n d 36+6 ~ Ci/~ g for BSA a n d Man-BSA. B - G l u e u r o n i d a s e a n d B - h e x o s a m i n i d a s e d e f i c i e n t h u m a n f i b r o b l a s t s w e r e g r o w n as d e s c r i b e d p r e v i o u s l y (2). P i n o c y t o s i s m e a s u r e m e n t s : The i n d i c a t e d p r o t e i n s w e r e i n c u b a t e d w i t h Con A (Sigma, g r a d e IV) 20 rain at room t e m p e r a t u r e i n the p r e s e n c e of 250 ~ g h u m a n s e r u m a l b u m i n , 0.15 M NaCl, 10 mM NazHPO 4, I0 mM T r i s - H C 1 , pH 7 . 6 , i n a f i n a l v o l u m e of 0.2 ml. One ml of E a g l e ' s Minimal E s s e n t i a l Medium c o n t a i n i n g 0.2% h u m a n s e r u m a l b u m i n was t h e n a d d e d a n d 1 ml of t h i s m i x t u r e was l a y e r e d onto c u l t u r e d f i b r o b l a s t s i n 35 mm p e t r i d i s h e s . The c e l l s w e r e a p p r o x i m a t e l y 75% c o n f l u e n t . After 1 h r i n c u b a t i o n at 37°C, d i s h e s w e r e c h i l l e d a n d w a s h e d s i x times w i t h cold p h o s p h a t e - b u f f e r e d s a l i n e . Cell a s s o c i a t e d a c t i v i t y was m e a s u r e d as d e s c r i b e d b y K a p l a n et a l . (2). ~ - G l u c u r o n i d a s e a n d h e x o s a m i n i d a s e w e r e a s s a y e d as d e s c r i b e d b y G l a s e r et al. (12) a n d p r o t e i n a c c o r d i n g to L o w r y et al. (13). R a d i o a c t i v i t y was m e a s u r e d i n a P a c k a r d gamma c o u n t e r . T h e r a t e of b i n d i n g a n d / o r u p t a k e is e x p r e s s e d as the p e r c e n t of a d d e d r a d i o a c t i v i t y or e n z y m a t i c a c t i v i t y w h i c h was cell a s s o c i a t e d p e r u n i t time (% u p t a k e / r a g protein/hr).
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60 CON Aj.o
_z I.d k0 40 ¢D
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CON _
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200
300
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F i g u r e 1. Cells w e r e w a s h e d with cold T r i s - p h o s p h a t e b u f f e r e d s a l i n e (TPBS) a n d t h e n i n c u b a t e d with 0.5 ml of TPBS c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s of ConAwith (•) or w i t h o u t ( O ) 50ram aMM. After 15 m i n a t 4°C d i s h e s w e r e w a s h e d a n d i n c u b a t e d with 1700 U of B - g l u e u r o n i d a s e in 0.5 ml of TPBS. At the e n d of the s e c o n d m e u b a t l o n perxod (15 mln at 4 C ) , p l a t e s w e r e w a s h e d a n d cell a s s o c i a t e d a c t i v i t y was m e a s u r e d .
RESULTS AND DISCUSSION: B i n d i n g of p l a c e n t a l ~ - g l u e u r o n i d a s e to Con A p r e e o a t e d cells - H u m a n p l a c e n t a l B - g l u e u r o n i d a s e w h i c h is p r e d o m i n a n t l y a " l o w - u p t a k e " form for f i b r o b l a s t s does nol b i n d d e t e e t a b l y to the cell s u r f a c e of h u m a n f i b r o b l a s t s . F i g u r e 1 s h o w s that p r i o r t r e a t m e n t of h u m a n f i b r o b l a s t s with Con A at 4°C a n d s u b s e q u e n t r e m o v a l of u n b o u n d l e e t i n b y e x t e n s i v e w a s h i n g , l e a d s to d r a m a t i c b i n d i n g of e n z y m e a d d e d to the f i b r o b l a s t s i n a s e c o n d i n c u b a t i o n at 4°C.
The a m o u n t of e n z y m e b o u n d is p r o p o r -
t i o n a l to the Con A c o n c e n t r a t i o n u s e d i n the p r i o r i n c u b a t i o n . E s s e n t i a l l y no e n zyme b i n d i n g o c c u r r e d w h e n 50 mM c~IM h a d b e e n p r e s e n t d u r i n g the e x p o s u r e of f i b r o b l a s t s to Con A.
T h e s e data s u g g e s t that the Con A s e r v e s as a b r i d g e b e t w e e n
cell s u r f a c e g l y e o p r o t e i n s a n d the " l o w - u p t a k e " ~ - g l u e u r o n i d a s e . Con A m e d i a t e d u p t a k e of " l o w - u p t a k e " B - g l u e u r o n i d a s e - It h a s b e e n p r e v i o u s l y e s t a b l i s h e d that Con A c a n b i n d to the s u r f a c e m e m b r a n e (14,15) a n d b e i n t e r n a l ized (16) b y f i b r o b l a s t s i n e u l t u r e .
H u m a n p l a c e n t a l B - g l u e u r o n i d a s e is i t s e l f ,
v e r y p o o r l y t a k e n u p b y h u m a n f i b r o b l a s t s (0.2% U / r a g / h ) .
H o w e v e r , m i x i n g the
e n z y m e w i t h free Con A i n a p r i o r i n c u b a t i o n a n d s u b s e q u e n t e x p o s u r e of cells to
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I00~ C ~
8O
.®
-~-MANNOSE 80 t \ \ -O'-MANNOSE-6-P ~-g- a- MET-
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'
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20
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F i g u r e 2. A. Con A m e d i a t e d b i n d i n g (4°C) a n d u p t a k e (37°C) of ~ - g l u c u r o n i d a s e . Cells i n 35 mm d i s h e s w e r e i n e u b a t e d for l h r at 4°C ( • ) a n d 37°C ( • ) with 1 ml of TPBS c o n t a i n i n g ~ - g l u c u r o n i d a s e (1700 U ) , Con A at the c o n c e n t r a t i o n s i n d i c a t e d a n d h u m a n s e r u m a l b u m i n (25 ~ g ) . T h e ~ - g l u e u r o n i d a s e a n d Con A h a d b e e n p r e i n e u b a t e d i n 0.2 ml of TPBS for 20 m i n at room t e m p e r a t u r e b e f o r e d i l u t i o n a n d a d d i n g to c e l l s . B. Time d e p e n d e n c e of Con A mediated e n z y m e u p t a k e . Con A (5 ~ g ) , p r e i n e u b a t e d with 1600 U of 8 - g l u c u r o n i d a s e was a d d e d to cells i n the p r e s e n c e of ( • ) or i n the a b s e n c e of ( • , • ) 50 mM c~IM. At the e n d of the i n c u b a t i o n p e r i o d cells w e r e w a s h e d with b u f f e r e d s a l i n e with ( • , • ) or w i t h o u t ( • ) 50 mM ~/M. C. I n h i b i t i o n of the Con A m e d i a t e d u p t a k e b y s u g a r s . Con A (5 g g) w a s p r e i n e u b a t e d with 1600 U of B - g l u c u r o n i d a s e a n d t h e n e x p o s e d to cells i n the p r e s e n c e of m a n n o s e ( • ) , m a n n o s e - 6 - p h o s p h a t e ( • ) or c~VIM ( • ) . Cell a s s o c i a t e d B - g l u e u r o n i d a s e was m e a s u r e d after a 1 h o u r i n c u b a t i o n at 37°C.
the e n z y m e - l e e t i n m i x t u r e p r o d u e e d an i n c r e a s e i n c e l l - a s s o c i a t e d e n z y m e .
Figure
2-A shows the effect of i n c r e a s i n g c o n c e n t r a t i o n s of Con A on the a m o u n t of c e l l - a s s o ciated e n z y m e w h e n the c e l l s w e r e e x p o s e d to the m i x t u r e s at 4°C a n d 37°C. At 4°C, w h e r e c e l l - a s s o c i a t e d e n z y m e p r e s u m a b l y r e p r e s e n t s e n z y m e b o u n d b u t not i n t e r n a l i z e d , maximum s t i m u l a t i o n of b i n d i n g o e e u r r e d at 5-8 ~ g / m l Con A.
Washing
the d i s h e s with PBS c o n t a i n i n g 50 mM ~MM after the i h o u r i n c u b a t i o n p e r i o d at 4°C, r e l e a s e d 29% of the c e l l - a s s o c i a t e d e n z y m e b o u n d with 5 g g / m l of Con A.
At 37°C
the l e c t i n p r o d u c e d a much l a r g e r i n c r e a s e i n c e l l - a s s o e i a t e d e n z y m e . A g a i n , the maximum s t i m u l a t i o n o e e u r r e d b e t w e e n 5-8 ~ g / m l ; b u t h i g h e r l e v e l s led to r e d u c e d uptake.
In t h i s e x p e r i m e n t , e n z y m e w h i c h b e c a m e c e l l - a s s o c i a t e d d u r i n g the 1 h o u r
i n c u b a t i o n at 37°C p r e s u m a b l y r e f l e c t e d the sum of e n z y m e b o u n d to the cell s u r face as a e o n s e q u e n c e of the i n t e r a c t i o n with the l e e t i n , a n d e n z y m e i n t e r n a l i z e d a f t e r
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binding.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In a g r e e m e n t with the e x p e c t a t i o n from this i n t e r p r e t a t i o n , o n l y 6% of the
e n z y m e t a k e n u p after 1 h o u r i n the p r e s e n c e of 5 la g / m l Con A was r e l e a s a b l e b y w a s h i n g with 50 mM aMM. Time d e p e n d e n c e of Con A m e d i a t e d ~ - g l u c u r o n i d a s e u p t a k e - F i g u r e 2-B s h o w s the time d e p e n d e n e e of Con A m e d i a t e d u p t a k e of p l a c e n t a l B - g l u c u r o n i d a s e at 37°C. T h e e n z y m e t a k e n u p , w h i c h i n c l u d e s the sum of b o u n d a n d i n t e r n a l i z e d e n z y m e , i n c r e a s e d l i n e a r l y u n d e r t h e s e c o n d i t i o n s for at least 60 rain. A d d i t i o n of aMM (50 mM), at the time the p r e s u m p t i v e e n z y m e - Con A complex was a d d e d to p l a t e s , l a r g e l y p r e v e n t e d the time d e p e n d e n t u p t a k e .
In c o n t r a s t , a d d i t i o n of the s u g a r
after the i n c u b a t i o n p e r i o d r e l e a s e d o n l y a small f r a c t i o n (6-17%) of the f i b r o b l a s t a s s o c i a t e d e n z y m e . T h i s o b s e r v a t i o n s u g g e s t e d that most of the cell a s s o c i a t e d e n zyme had b e e n i n t e r n a l i z e d d u r i n g the i n c u b a t i o n at 37°C. I n h i b i t i o n of the Con A m e d i a t e d u p t a k e b y s i m p l e s u g a r s - F i g u r e 2-C c o m p a r e s the i n h i b i t i o n of p l a c e n t a l ~ - g l u c u r o n i d a s e u p t a k e b y s u g a r s p r e s e n t i n the m e d i u m to w h i c h the Con A / B - g l u c u r o n i d a s e m i x t u r e was a d d e d ,
aMM was c l e a r l y the most
p o t e n t i n h i b i t o r , about 5 fold more p o t e n t t h a n the o t h e r s u g a r s b a s e d on the concent r a t i o n p r o d u c i n g half of maximum i n h i b i t i o n . T h e s e r e s u l t s c o n t r a s t s h a r p l y with the r e s u l t s of t h e s e i n h i b i t o r s on the a d s o r p t i v e p i n o e y t o s i s of acid h y d r o l a s e s b y the p h o s p h o m a n n o s y l r e c o g n i t i o n s y s t e m i n f i b r o b l a s t s . T h e r e , m a n n o s e ~ 6 - P i s about 1,000 times more potent t h a n m a n n o s e or aMM as an i n h i b i t o r of e n z y m e p i n o c y t o s i s (2). Effect of Con A on the u p t a k e of o t h e r p r o t e i n s - E d e l s o n a n d Cohn (17) r e p o r t e d that Con A p r o d u c e d a four fold e n h a n c e m e n t of u p t a k e of h o r s e r a d i s h p e r o x i d a s e (HRP) b y mouse p e r i t o n e a l m a e r o p h a g e s .
T h e y did not a t t r i b u t e this s t i m u l a t i o n to
the i n t e r a c t i o n b e t w e e n Con A a n d the m a n n o s e - r i c h g l y e o p r o t e i n (HRP), s i n c e they o b s e r v e d s i m i l a r s t i m u l a t i o n of [1251] BSA u p t a k e . We h a v e e x a m i n e d the effects of Con A on two p r o t e i n s w h i c h c o n t a i n no c a r b o h y d r a t e , a n d also on s e v e r a l g l y e o p r o t e i n s . In F i g . 3 we c o m p a r e the s t i m u l a t i o n of u p t a k e b y Con A for [125I] - m a n n o s e - B S A , a n d [1251]RNaseB.
RNaseBhas
a s i n g l e o l i g o s a e c h a r i d e c h a i n a n d is q u a n t i t a t i v e l y r e t a i n e d on Con A - S e p h a r o s e
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I,G o~ -r ¢lJ
0.8 ~
co.= O E
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9
40
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F i g u r e 3. Effect of Con A on u p t a k e of g l y c o s y l a t e d a n d n o n - g l y c o s y l a t e d p r o t e i n s . E x p e r i m e n t s w e r e p e r f o r m e d a s d e s c r i b e d in F i g . 2, Con A w a s p r e i n c u b a t e d in the p r e s e n c e of 5 p m o l e s o f [ 1 2 5 I ] B S A ( A ) , [ 1 2 5 1 ] M a n - B S A (•), 125I]RNaseA ( O ) o r [ I25 I]RNaseB ( • ), before the mlxturewas added to t h e d i s h e s . Cell a s s o c i a t e d r a d i o a c t i v i t y m e a s u r e d a f t e r a 1 h o u r i n c u b a t i o n at 37°C. columns.
r
3
O n l y 16% of the L125ij Man-BSA c o n t a i n i n g an a v e r a g e of 20 m a n n o s e s u b -
s t i t u t i o n s was r e t a i n e d b y Con A - S e p h a r o s e a n d s p e c i f i c a l l y e l u t e d .
S t i m u l a t i o n of
u p t a k e b y Con A i s s e e n b o t h w i t h [1251] RNase B a n d [1251] M a n - B S A , b u t not w i t h t h e c o r r e s p o n d i n g n o n c a r b o h y d r a t e c o n t a i n i n g p r o t e i n s ( F i g . 3).
Note t h a t t h e
s t i m u l a t i o n s e e n w i t h t h e s e g l y e o p r o t e i n s is v e r y s m a l l c o m p a r e d to t h a t for ~ - g l u c u r o n i d a s e ( F i g . 2) o r I - c e l l s e c r e t i o n h e x o s a m i n i d a s e ( F i g . 4 ) .
Figure 4 (A-C)
p r e s e n t s e x p e r i m e n t s s h o w i n g the effect of Con A on t h e u p t a k e of t h r e e d i f f e r e n t g l y c o p r o t e i n s , RNase B, h u m a n p l a c e n t a l ~ - g l u c u r o n i d a s e , a n d h e x o s a m i n i d a s e from I - c e l l s e c r e t i o n s . w e l l c h a r a c t e r i z e d (18).
T h e s i n g l e o l i g o s a c c h a r i d e c h a i n of RNase B h a s b e e n Human p l a c e n t a l ~ - g l u c u r o n i d a s e i s a t e t r a m e r w h i c h c o n -
t a i n s 6.5% c a r b o h y d r a t e s , a n d is m a n n o s e - r i e h (19). s a c c h a r i d e c h a i n s p e r m o n o m e r i s not k n o w n .
H o w e v e r , the n u m b e r of oligo-
Little o r n o t h i n g i s k n o w n of t h e
c a r b o h y d r a t e c h a i n c o m p o s i t i o n of I - c e l l s e c r e t i o n h e x o s a m i n i d a s e .
H o w e v e r , it i s
q u a n t i t a t i v e l y a d s o r b e d from c u l t u r e m e d i u m b y Con A - S e p h a r o s e (A. G o n z a l e z N o r i e g a a n d W. S. S l y , u n p u b l i s h e d o b s e r v a t i o n s ) .
The stimulation, by increasing
c o n c e n t r a t i o n s of Con A , of u p t a k e of t h e s e t h r e e d i f f e r e n t g l y c o p r o t e i n s , s t u d i e d e i t h e r i n d i v i d u a l l y o r in t h e p r e s e n c e of a s e c o n d g l y c o p r o t e i n , i s s h o w n in F i g . 4. T h e p a t t e r n of s t i m u l a t i o n a p p e a r e d d i s t i n c t i v e for e a c h of t h e t h r e e g l y c o p r o t e i n s studied.
A d d i t i o n of a s e c o n d g l y c o p r o t e i n i n f l u e n c e d the l e v e l of s t i m u l a t i o n s e e n
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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
14
~ RNase B ALONE
/
LOCU"ON'OASE i I"I0
rr
I0 Z
-®
W I-0
HEXOSAMINIDASE ALONE -m- + ,8-GLUCURONIDASE + RNase B
L9 W < I-n
I
I
28 4 - /9-GLUCURONIDASE ALONE --o- + HEXOSAMINIDASE se B
20
12 e
5'o
•
,~o
,~o
26o
a~o
CON A (p.G/ML)
F i g u r e 4. P e r c e n t of cell a s s o c i a t e d [ 1251] RNase B (A), h e x o s a m i n i d a s e (B) a n d ~ - g l u c u r o n i d a s e (C) w h e n the i n d i c a t e d c o n c e n t r a t i o n of Con A was p r e i n c u b a t e d with each g l y c o p r o t e i n s i n g l y or with one of the o t h e r two g l y c o p r o t e i n s . Amount of each a d d e d w h e n p r e s e n t : [1251] RNase B (5 p m o l e s ) , h e x o s a m i n i d a s e (1300 U) B - g l u c u r o n i d a s e (1600 U ) . Cell a s s o c i a t e d e n z y m e a c t i v i t y or r a d i o a c t l v l t y w e r e m e a s u r e d after a 1 h o u r x n c u b a t l o n at 37 C.
(in all 3 c a s e s e n h a n c i n g u p t a k e ) , b u t did not a p p e a r to a l t e r the p a t t e r n of u p t a k e of the f i r s t l i g a n d .
We h a v e no e x p l a n a t i o n for t h i s effect.
Con A is k n o w n to b e a t e t r a v a l e n t l e c t i n (20).
As s u c h , it might b e e x p e c t e d
to s e r v e as a c r o s s - l i n k i n g a g e n t b e t w e e n cell s u r f a c e g l y c o p r o t e i n s a n d some glyco p r o t e i n s i n the m e d i u m .
S u c h r e a c t i o n s c o u l d b e q u i t e complicated s i n c e e i t h e r the
cell s u r f a c e g l y c o p r o t e i n or the p r o t e i n i n the medium c a n b e m o n o v a l e n t or m u l t i -
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valent itself.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
P r e s u m a b l y s u c h d i f f e r e n c e s i n the e n z y m e s e x p l a i n s the d i f f e r e n t
p a t t e r n s of s t i m u l a t i o n b y Con A. Beeck et a l . (21) r e p o r t e d an a b s t r a c t on the effects of Con A a n d s u e c i n y l Con A on e n d o c y t o s i s of l y s o s o m a l e n z y m e s . T h e i r s t u d i e s d i f f e r e d from those p r e s e n t e d here.
The e n z y m e s u s e d w e r e s u b j e c t to a d s o r p t i v e e n d o c y t o s i s i n the a b s e n c e of
l e c t i n ( i . e . w e r e " h i g h u p t a k e " forms) a n d t h e y d e s c r i b e d i n h i b i t i o n of e n z y m e p i n o c y t o s i s b y the l e c t i n s .
T h e s e effects w e r e a t t r i b u t e d to i n t e r a c t i o n s of the l e c t i n with
both the r e c o g n i t i o n m a r k e r on the e n z y m e s a n d the f i b r o b l a s t r e c e p t o r .
We i n t e n -
t i o n a l l y r e s t r i c t e d o u r s t u d i e s to the effects of Con A on p i n o c y t o s i s of g l y c o p r o t e i n s w h i c h are not s u b j e c t to a d s o r p t i v e e n d o c y t o s i s . O u r s t u d i e s d e m o n s t r a t e d d r a m a t i c s t i m u l a t i o n of p i n o c y t o s i s of t h e s e g l y c o p r o t e i n s b y Con A u n d e r c o n d i t i o n s w h e r e u p t a k e of n o n - g l y c o p r o t e i n s was not affected.
We i n t e r p r e t t h e s e o b s e r v a t i o n s to
i n d i c a t e that the l e c t i n can s e r v e as a b r i d g e b e t w e e n a l e e t i n b i n d i n g site on the cell s u r f a c e a n d a l e c t i n b i n d i n g site on the g l y c o p r o t e i n a n d lead to u p t a k e of the glycoprotein by "piggyback endoeytosis." The f i n d i n g that Con A c a n mediate u p t a k e of a g l y c o p r o t e i n o t h e r w i s e not s u s c e p t i b l e to a d s o r p t i v e p i n o e y t o s i s has i m p o r t a n t p r a c t i c a l i m p l i c a t i o n s . Many g l y c o p r o t e i n s a r e i s o l a t e d b y affinity c h r o m a t o g r a p h y u s i n g Con A - S e p h a r o s e .
S i n c e free
Con A is k n o w n to come off s u c h c o l u m n s (8), s t u d i e s of e n z y m e u p t a k e u s i n g e n zymes i s o l a t e d on Con A - S e p h a r o s e c o l u m n s must be i n t e r p r e t e d v e r y c a u t i o u s l y . One must p r o v i d e e v i d e n c e that no free Con A is p r e s e n t i n the e n z y m e p r e p a r a t i o n , or that the u p t a k e is not i n h i b i t e d b y ~MM. A p r a c t i c a l w a y to d i s t i n g u i s h Con A m e d i a t e d u p t a k e b y f i b r o b l a s t s is to i n c l u d e 25 mM ~//M i n the i n c u b a t i o n . T h a t c o n c e n t r a t i o n is too low to i n h i b i t the u p t a k e b y the 6 - p h o s p h o m a n n o s y l r e c o g n i t i o n s y s t e m (2) b u t effectively b l o c k s the Con A m e d i a t e d u p t a k e w h i c h we h a v e d e s c r i b e d here.
ACKNOWLEDGEMENTS: T h i s w o r k is s u p p o r t e d i n p a r t b y the U . S . P . H . S . g r a n t 21096 a n d the R a n k e n J o r d a n F u n d for the C r i p p l i n g D i s e a s e s of C h i l d r e n . T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e the a d v i c e a n d a s s i s t a n c e of D r . D a n i e l A e h o r d , D a v i d F i s c h e r , a n d M a r v i n Natowicz on s e v e r a l a s p e c t s of t h i s w o r k , a n d D r s . R o s a l i n d K o r n f e l d , Paul S c h l e s i n g e r , a n d P h i l i p Stahl for c r i t i c i s m s of the manuscript.
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