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Study of nuclear mannosyl-transferase : lipids intermediates
M6moires originaux
BIOCHIMIE, 1978, 60, 5'9'3-599.
Study of nuclear mannosyl-transferase : lipids intermediates. M. R I C H A R D (*), F. T Y T G A T a n d P. L O U I S O T ~.
University o[ Lyon, Lyon-Sud Medical School, Laboratory o[ B i o c h e m i s t r y and ERA-CNBS, 562, 69600 Oullins, France.
R6sum6.
Summary.
La m m m o s y l - t r a n s f 6 r a s e de n o y a u de foie de rat c a t a l y s e le transfert du m a n n o s e , & partir du G D P - m a n n o s e , sur des a c c e p t e u r s e n d o q ~ n e s lipidiques o u prot6iniques. Deux q l y c o l i p i d e s d e solubilit6s diff6rentes ont 6t6 caract6ris6s : un m a n n o s y l - p h o s p h o r y l - d o l i c h o l et un oliqosaccharide-lipide. Le m a r q u a q e radioactif de ces deux lipides est c o m p a t i b l e a v e c le r61e qu'ils p e u v e n t jouer d a n s le transfert du m a n n o s e a u x prot6ines nucl6aires, m a i s p a s s e l o n le m6can i s m e de Lennarz.
M a n n o s y l - t r a n s f e r a s e of rat liver nuclei catal y z e d the transfer of m a n n o s e , from GDP-mann o s e , to e n d o q e n o u s lipids a n d proteins. T w o solubility-different qlycolipids w e r e characterized : a mannosyl-phosphoryl-dolichol and an oliqosaccharide-lipid. The labelinq of the two lipids w a s consistent with a role in m a n n o s e transfer to n u c l e a r qlycoproteins, but not accordinq to the Lennarz' s c h e m e .
Introduction.
Material and Methods.
I n t h e l a s t t h r e e y e a r s , m a n y w o r k s [1-7] h a v e shown the part taken by polyprenic compounds i n t h e b i n d i n g of o l i g o s a c c h a r i d e c h a i n s t o g l y c o proteins. These works study chiefly the incorpor a t i o n of m a n n o s e a n d N - a c e t y l - g l u c o s a m i n e b y membrane-bound enzymatic systems. P r e v i o u s p a p e r s [8-10] r e p o r t e d g l y c o s y l - t r a n s f e r a s e l o c a l i z a t i o n i n a n u c l e a r cell f r a c t i o n . I n t h e r a t l i v e r n u c l e i [10] e v i d e n c e w a s g i v e n f o r t h e e x i s t e n c e of f i v e g l y c o s y l - t r a n s f e r a s e s : m a n n o s y l - , g a l a c t o s y l - , N - a c e t y l - g l u c o s a m i n y l - , Na c e t y l - g a l a c t o s a m i n y l - a n d s i a l y l - t r a n s f e r a s e . So it s e e m s i n t e r e s t i n g to s t u d y t h e p l a c e of p o l y p r e n i c compounds in nuclear glycoprotein biosynthesis. This paper reports data obtained in such a study, c o n c e r n i n g m a n n o s e b i n d i n g to o l i g o s a c c h a r i d e c h a i n of g l y c o p r o t e i n s .
( * ) Altach6 de R e c h e r c h e s au CNRS.
To whom all correspondence should be addressed.
Key words : mannosyl-transferase, nuclei, glycoproteins, glycolipids, glycosyl-transfera~e.
- - Nuclear enzyme preparation. Rat liver nuclei were prepared according to the Zalta m e t h o d [11] a d a p t e d by Beck el al. [12I. The p u r e n e s s of the nuclear f r a c t i o n was checked by elect r o n microscopy and m a r k e r enzymes [10].
- - A s s a y for incorporation of [l~C]mannose from GDP-[l~C]mannose into endogenous aceepfors. The s t a n d a r d assay m i x t u r e to s t u d y the incorporat i o n of [14C]mannose from GDP-[14C]mannose into the t h r e e endogenous acceptors c o n t a i n e d 0.2' ml of nuclear suspension (1 mg of protein) in 0.05 M Trism a l e a t e buffer pH ~ 6,, 0.25 M sucrose, 0.01 ml of 0.11 M MnCl~ and 0.01 ml of a solution c o n t a i n i n g 117 picomoles of G*DP-[14C]mannose (specific activity 170 Ci/M, NEN Chemicals USA). The r e a c t i o n was started by the a d d i t i o n of GDP-E14C]mannose and carried out at 30°C. The used e x t r a c t i o n procedure was described by W a e c h t e r et al. [13]. The r e a c t i o n was stopped at the indicated t i m e b y the a d d i t i o n of 4.4 ml of CHCI~ - CH3OH (2:1). The m i x t u r e was allowed to s t a n d at room t e m p e r a t u r e for 15' m i n u t e s and t h e n centrifuged. The s u p e r n a t e was removed and the pellet was extracted in the same m a n n e r . The c o m b i n e d CH,CI.~ - - CH,~OH : extracts were t h e n w a s h e d w i t h 2.5 ml of 0.9 per cent NaC1. The upper phase was discarded a n d the lower phase w a s h e d w i t h 3 ml of 0.9 per cent NaCl - - CH, OH (1:0.5). The dried pellet from the CHCI~ ~ CHa0H
594
M. Richard, F. Tytgat and P. Louisot.
e x t r a c t i o n w a s w a s h e d t h r e e t i m e s w i t h 2 m l o f H20 ; the washes were discarded. The pellet was then extract e d t w i c e in 5 m l o f CHCI~ - - CH~OH - - H ~ (1:1:0.3). The residue was conserved. The assay method separated the three [14C]mannose labeled endogenous acceptors : mannolipid, which was s o l u b l e i n C ~ C l z - C H ~ O H (2:1) ; m a n n o s y l - S - a c e e p t o r w h i c h w a s s o l u b l e i n C~CIs - CHzOH, - H~O (1:1:0.3) ; m a n n o s y l - r - a c c e p t o r w h i c h w a s i n s o l u b l e in b o t h of t h e a b o v e s o l v e n t s a n d i n w a t e r ( a c c o r d i n g to L e n n a r z ' s n o m e n c l a t u r e [13]). - - P a r t i a l p u r i f i c a t i o n of [~.',C]mannolipid. T h e o r g a n i c p h a s e f r o m t h e CI~Clz-CHzOH (2:1) e x t r a c t i o n w a s e v a p o r a t e d to d r y n e s s u n d e r r e d u c e d p r e s s u r e . T h e c r u d e l i p i d w a s r e d i s s o l v e d i n CH'CI~CH~O~ (7:3)~ a n d a p p l i e d to a D E A E - c e l l u l o s c c o l u m n (3 X 28 cm) t h a t h a d b e e n e q u i l i b r a t e d i n CHClaCH~OH (7:3). T h e c o l u m n w a s s e q u e n t i a l l y e l u t e d w i t h 10'00 m l of CHCI3 - CH,OIt ( 7 : 3 ) a n d 500 m l of CH~OH. A s m a l l p e r c e n t a g e of t h e r a d i o a c t i v i t y ( a p p r o x i m a t e l y 1.5 p e r c e n t ) w a s r e c o v e r e d i n t h e s e f r a c t i o n s . T h e c o l u m n w a s t h e n e l u t e d w i t h 0.q2 ~ a m m o n i u m acet a t e i n 99 p e r c e n t CH~OH. ~5 p e r c e n t of t h e r a d i o a c t i vity was recovered in a single peak. The fractions cont a i n i n g r a d i o a c t i v i t y w e r e p o o l e d a n d a d d e d to 2 v o l u m e s of CHCI~. T h e m i x t u r e w a s w a s h e d w i t h 1/5 v o l u m e of H20 to r e m o v e a m m o n i u m a c e t a t e . T h e o r g a n i c p h a s e w a s e v a p o r a t e d to d r y n e s s u n d e r r e d u c e d p r e s s u r e a n d d i s s o l v e d i n 3 m l of CI-I~OH - t o l u e n e (1:1) a n d t r e a t e d w i t h a l k a l i [14]. T h e d e a c y l a t e d l i p i d f r a c t i o n ( a p p r o x i m a t e l y 80 p e r c e n t of t h e r a d i o a c t i v i t y ) w a s a p p l i e d to a n o t h e r DEAE-cellulose column (2 X 15 era) e q u i l i b r a t e d in CI-IEI~- CH~OH (7~:3). T h e c o l u m n w a s e l u t e d w i t h 109 m l o f GH,CI~- CH~OH. (7:3), 100 m l o f CH:~OH a n d a c o n t i n u o u s g r a d i e n t of a m m o n i u m a c e t a t e (0-0:03 M9 i n m e t h a n o l . 7~ p e r c e n t of t h e r a d i o a c t i v i t y w e r e e l u t e d in a s i n g l e s y m m e t r i c a l peak with the continuous gradient. Ammonium acetate w a s r e m o v e d as p r e v i o u s l y d e s c r i b e d . T h e l i p i d w a s s t o r e d a t - - 20°C. - - P a r t i a l p u r i f i c a t i o n of m a n n o s y l - S - a c c e p t o r . T h e C~C13 - CH3OH - H~O (1:1:0.3) extracts were a p p l i e d to a DEA~E-ccllulose C o l u m n (3 X 25 c m ) , t h a t h a d b e e n e q u i l i b r a t e d i n CHCI~ - CH~OH - H~O (1:1:0.3). T h e c o l u m n w a s w a s h e d w i t h 500 m l of CHCla - CHaOH H~O (1:1:0.3), (no r a d i o a c t i v i t y w a s r e c o v e r e d in t h i s f r a c t i o n ) , t h e n c l u t e d w i t h 0.1 M a m m o n i u m a c e t a t e i n CHCI~- CH~Otf- H~O (1:1:0:3). I n t e r m e d i a t e s t e p s w i t h l o w e r s a l t c o n c e n t r a t i o n s e l u t e d 5 p e r c e n t of t h e radioactivity. The labeled fractions were pooled, conc e n t r a t e d , w a s h e d to r e m o v e a m m o n i u m a c e t a t e a n d s t o r e d at - - 2 0 ° C . -- Chromatographic procedures. Partially purified mannolipid was thin-layer chrom a t o g r a p h e d o n s i l i c a gel. T h e d e v e l o p i n g s o l v e n t s w e r e : A, C~CI~ - CH~OH - CH~COOH - H~O (26~:16:14:2) ; B, C H C I ~ - C H ~ O H - N H ~ O H (75:25:4) : C. n - p r o p a n o l H~O (8:2). T h e p o l y p r e n o l s w e r e d e t e c t e d b y e x p o s i n g t h e c h r o m a t o g r a m s to a n i s a l d e h y d [15]. l ~ a n n o s y l - S - a c c e p t o r w a s c h r o m a t o g r a p h e d o n silica gel w i t h s o l v e n t s A, B, C, D, n - p r o p a n o l - H~O'(7:3) a n d E, n - p r . o p a n o l - H~O (5:3). GDP-mannose, mannose-phosphate and mannose w e r e s e p a r a t e d o n W h a t m a n N ° 3 MM: p a p e r in t h e s o l v e n t F , e t h a n o l - M CH~COO N : a p H ; = 3 . 6 (75:30), B ! O C H I M I E , 1978, 60, .u ° 6-7.:
mannose-l-phosphate and mannose-6-phosphate on E c t e o l a - - c e l l u l o s e i n t h e s o l v e n t G, 95 p e r c e n t e t h a nol --0.1 M a m m o n i u m t e t r a b o r a t e pIff = 9 (3:2% These compounds were detected by exposing the chrom a t o g r a m s to p e r i o d a t e - b e n z i d i n c [16]. N e u t r a l s u g a r s w e r e c h r o m a t o g r a p h e d o n s i l i c a gel i n t h e s o l v e n t H, l ' - b u t a n o l - p y r i d i n e - 0 . 1 N HE1 (5:3:2). S t a n d a r d s u g a r s w e r e l o c a t e d b y a n i l i n e phtalate. -- Hydrolytic procedures. C o n d i t i o n s f o r acid h y d r o l y s i s of m a n n o l i p i d a r e described in the figure legend. Alkaline hydrolysis was p e r f o r m e d b y 0.1 N N a O H in 90 p e r c e n t e t h a n o l at 80°C f o r 15 m i n u t e s . D i f f e r e n t c o m p o u n d s w e r e e x t r a c t e d b y t h e F o l c h ' s m e t h o d [17]. W a t e r - s o l u b l e p r o d u c t s w e r e e h r o m a t o g r a p h e d o n ,l~cteola-ccllulose i n t h e solv e n t G. M a n n o s y l - S - a e c e p t o r s , p r e p a r e d f r o m GI)P-[14C] m a n n o s e or [ 1 4 C ] m a n n o l i p i d , m a n n o s y l - r - a e c e p t o r s a n d X c o m p o u n d s (see r e s u l t s ) w e r e t r e a t e d b y 3N HC1 at l(r0°C f o r 3 h o u r s . H y d r o l y s a t e s w e r e f r a c t i o n a t e d on D o w e x 51) X 4 (200-400 m e s h ) a n d D o w e x 1 × 8 (200400 m e s h ) . C o n c e n t r a t e d e l u a t e s w e r e c h r o m a t o g r a p h e d o n silica gel i n t h e s o l v e n t H. -- Enzyme digestion. Mannosyl-r-aeeeptor prepared from GDP-mannose a n d m a n n o s y l - s - a c e e p t o r w a s i n c u b a t e d i n 5 m l of 0.05 M Tris-H)C1 p ~ = 8, 0.01 m l of M, CaCl~, 1 m g of p r o n a s e . A d r o p of t o l u c n w a s a d d e d a n d t h e r e a c t i o n w a s c a r r i e d o u t a t 37°C. A d d i t i o n a l p r o n a s c (0.5 rag) w a s a d d e d a t 5 a n d 24 h o u r s . At 48 h o u r s t h e m i x t u r e w a s f r o z e n f o r l a t e r use. X c o m p o u n d (see r e s u l t s ) w a s i n c u b a t e d w i t h a l k a l i n e p h o s p h a t a s e ( W o r t h i n g t o n ] b i o c h e m i c a l Corp.) i n 0.1 M Tris-HC1, p H 7.8, a t 37°C f o r 3 h o u r s or w i t h a - m a n n o s i d a s e ( B o e h r i n g e r ) in 0.5 M s o d i u m c i t r a t e , p H = 4.5 at 37°C f o r 36 h o u r s . - - Gel f i l t r a t i o n . A S e p h a d e x G-50 c o l u m n (2.5)< 50 em), w a s p r e p a r e d a n d e q u i l i b r a t e d w i t h 0.06 M Tris-HC1, p H : 7.2, c o n t a i n i n g 0.5 p e r c e n t s o d i u m d e o x y e h o l a t e . T h e c o l u m n was calibrated with proteins, baeitraein and sucrose. U n t r e a t e d or p r o n a s e - t r e a t e d m a n n o s y l - r - a e e e p t o r s , m a n n o s y l - s - a e e e p t o r a n d X - c o m p o u n d w e r e a p p l i e d to the column. -- Paper electrophoresis. X c o m p o u n d s (see r e s u l t s ) w e r e a p p l i e d to W h a t m a n No 3 MM p a p e r a n d s u b j e c t e d to e l e e t r o p h o r e s i s (40 v o l t s p e r e m f o r 1.5 h o u r s ) w i t h p y r i d i n e - CH~COOHH~O (pH : 6.5) or 1 p e r c e n t s o d i u m t e t r a b o r a t e , p H = 9.5. - - A s s a y f o r i n c o r p o r a t i o n of [ l ' , C ] m a n n o l i p i d a n d m a n n o s y l - S - a c c e p t o r into e n d o g e n o u s acceptors. P a r t i a l l y p u r i f i e d m a u n o l i p i d , e v a p o r a t e d to d r y n e s s u n d e r r e d u c e d p r e s s u r e , w a s d i s s o l v e d in a 5 p e r c e n t T r i t o n X-l,00 s o l u t i o n (in t h i s d e t e r g e n t t h e d i s . s o l u t i o n w a s c o m p l e t e ) . 0.02 m l w e r e a d d e d to 0.2 m l of n u c l e a r s u s p e n s i o n (1-1.5 m g p r o t e i n ) w i t h or w i t h o u t Mn2+. F i n a l c o n c e n t r a t i o n of T r i t o n X-100 w a s 0.5 p e r cent. D i f f e r e n t c o m p o u n d s w e r e e x t r a c t e d as d e s c r i b e d a b o v e . M a u n o s y l - S - a e c e p t o r w a s i n t r o d u c e d in t h e s y s t e m b y s u b s t i t u t i o n of T r i t o n X-100 w i t h 5 p e r c e n t s o d i u m
Nuclear mannosyl-transferase deoxycholate. method.
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--- Routine methods. P r o t e i n w a s d e t e r m i n e d b y the m e t h o d of Lowry. Radioactivity was m e a s u r e d on a P a c k a r d Tri-Carb liquid s c i n t i l l a t i o n counter.
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1 °) K i n e t i c s o[ [ l ~ C ] m a n n o s e f r o m GDP-[1~C]mannose.
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incorporation
T h e t i m e c o u r s e of i n c o r p o r a t i o n of m a n n o s e from GDP-[~4C]mannose into three endogenous a c c e p t o r s i n t h e n u c l e a r f r a c t i o n is s h o w n i n E
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F]6. 2. - - 2a : melal ion requirement for mannolipid synthesis. 2b : dependence on pH of mannolipid synthesis. 2 e : dependence on temperature of mannolipid synthesis. 2d : dependence on GDP-mannose concentration of mannolipid synthesis.
i' 'o
1~
3'0
~'o
minutes FIG. 1 . Time course of [l$C]mannose transfer from GDP-[I~C] mannose into rat liver nuclei. Mannolipid, S-aeeeptor a n d r-aeeeptor were obtained as deseribed in Material a n d Methods.
3"
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f i g u r e 1. T h e s e t h r e e p r o d u c t s w e r e d e f i n e d b y their solubility properties. The mannolipid is s o l u b l e i n CHC13 -CaX-IaOH (2:1), m a n n o s y l - S - a c c e p t o r is s o l u b l e i n CHC13 - CH3OH - H 2 0 (1,:1:0.3) a n d m a n n o s y l - r - a c c e p t o r is f o u n d i n t h e i n s o l u b l e p e l let. It is e v i d e n t f r o m figure 1 t h a t l a b e l e d m a n n o l i p i d is f o r m e d at a m u c h g r e a t e r r a t e ( × 10) a n d at h i g h e r l e v e l s t h a n m a n n o s y l - S - a c c e p t o r a n d , after 7 minutes, undergoes turnover. Mannosyl-ra c c e p t o r is f o r m e d m o r e s l o w l y a n d is m e t a b o l i c a l l y s t a b l e . D e s i d e s , it is n o t e w o r t h y t h a t e x i s t s BIOCHIMIE, 1978, 60, n ° 6-7.
El
'
2'0
4'0
'
6'0 minutes
Fro. 3. - - Mild acid hydrolysis of mannolipid. The reaction was carried out at 35°C in 2.5 ml of 0.1 N H,CI in CH~OH. At the indicated times, 0.1 ml was w i t h d r a w n a n d t r a n s f e r r e d to a m i x t u r e of 2 ml of CHCI.~CH~Ott (2:1) a n d 0.5 ml of 0.1)2 N NaOH - - The m i x t u r e was mixed a n d the r a d i o a c t i v i t y was m e a s u r e d in the aqueous a n d organic phases.
M. Richard, F. T y t g a t and P. Louisot.
596
a p r e c u r s o r - d e r i v a t i v e relation a c c o r d i n g to Zilv e r s m i t [183 b e t w e e n S-acceptor and r-acceptor. 2 °) Conditions [or synthesis of mannolipid.
It was i n t e r e s t i n g to verify the reversibility of this enzymatic reaction. E x p e r i m e n t s were carried out on a n u c l e a r s u s p e n s i o n c o n t a i n i n g preformed, endogenous [14CJmannolipid w i t h diffe-
The t r a n s f e r of [l~C]mannose from C~D'P-[z4C] m a n n o s e into m a n n o l i p i d (fig 2a) requires a divalent metal ion, Mn 2÷ being nlore effective t h a n Mg 2÷. The optimal c o n c e n t r a t i o n of Mn 2÷ is approximately 0.005 M. The optimal pH for m a n n o l i p i d synthesis is about 6 (fig. 2b), the optimal temperature about 30°C (fig. 2c)'. F r o m the d e p e n d e n c e of the f o r m a t i o n of labeled m a n n o l i p i d on GD~P-mannose c o n c e n t r a t i o n s h o w n in figure 2d, an appar e n t K M for GDP-mannose of 0.6 t~M is calculated. 3 °) Properties of partially purified mannolipid. a) T r e a t m e n t of m a n n o l i p i d w i t h alkali a n d acid. T r e a t m e n t of the p a r t i a l l y p u r i f i e d m a n n o l i p i d w i t h 0.1 N HC1 at 35°C resulted in a r a p i d release of free mannose. The k i n e t i c of h y d r o l y s i s s h o w n in figure 3 indicates that the t 1/2 for the m a n n o lipid is 15 minutes. Mannose was the only p r o d u c t formed. The m a n n o l i p i d was stable to m i l d alkaline methanolysis (0.1 N NaOH, at 35°C for 60 minutes). A water-soluble p r o d u c t was released by strong alkaline h y d r o l y s i s (0.1 N NaOH in 90 per cent ethanol at 80°C for 16 minutes). The water-soluble product had a c h r o m a t o g r a p h i c m o b i l i t y s i m i l a r to that of m a u n o s e - l - p h o s p h a t e on Ecteola-cellulose in solvent system 6. b) C h r o m a t o g r a p h i c properties of m a n n o l i p i d . T h i n - l a y e r c h r o m a t o g r a p h i e s of partially purified [ l ~ C ] m a n n o l i p i d , on silica gel, in solvents A, B, C, exhibited R r, respectively 0.Y5, 0.40 and 0.78. W i t h these three solvent mixtures, [~*C]mannol i p i d was c h r o m a t o g r a p h i c a l l y identical to a u t h e n tic [a4C]mannosyl-phosphoryl-dolichol p r e p a r e d by Dr. Hemming.
4 °) Mannolipid as a precursor of mannosyl-Sacceptor. Exogenous p a r t i a l l y p u r i f i e d [14C]mannolipid was tested as a m a n n o s e donor. The results s h o w n in figure ~ reveal that n u c l e a r fraction catalyzed the t r a n s f e r of [14C]mannose from exogenous m a n n o l i p i d into mannosyl-S-acceptor. No watersoluble p r o d u c t and no r-acceptor was detected. This r e a c t i o n did not r e q u i r e Mn 2+. 5 °) Effects o[ nucleotides on endogenous man-
nolipid. M a n n o l i p i d is synthesized a c c o r d i n g to the reaction s h o w n below : L i p i d -4- ~ D P - m a u n o s e Mn2+ m a n n o l i p i d + GDP
BIOCHIMIE, 1978, 60, n ° 6-7.
-
1.5
t,.
,--
t~ £2
.s
tO
= /
~.racceptor,aqueous phase. 15
FIG. 4.
exogenous
-
-
30
fi'O
minutes. Synthesis of mannosyl S-acceptor from [l~C]mannolipid. Incubation conditions
were described in Material and Methods.
rent nucleotides, w i t h or w i t h o u t Mn 2~. GDP-[14C] m a n n o s e absence of r e i n c u b a t i o n m e d i u m was verified by high voltage electrophoresis. F r o m the tables I and H, it appears : a d i m i n u t i o n of the r a d i o a c t i v i t y i n the CHC13 -CH3OH (2:1) extracts w i t h di-and triphosphate nucleosides. Monophosphate-nucleosides, 3'-5' cyclic AMP and n o n h y d r o l y s a b l e nucleotides had no effect. - no GDP-[l~C]mannose at the end of the reinc u b a t i o n period. - -
- - an increase of the r a d i o a c t i v i t y in the CHC13 CHaOH- H20 (1:1:0.3) extracts with di-and triphosphate-nueleosides. - - an increase of the r a d i o a c t i v i t y in the r-aceeptor w i t h d i - a n d t r i p h o s p h a t e - n u c l e o s i d e s and Mn 2+. I n view of these results that are not in favour of the r e v e r s i b i l i t y of m a n n o l i p i d synthesis, we have used a different method. Results o b t a i n e d by D a l l n e r et al [19] have s h o w n that nuclei have a very high c o n c e n t r a t i o n of dolichol-phosphate, that w o u l d p r e v e n t the r e v e r s i b i l i t y of t h e reac-
0
0
200
300
2200
Without nucleotide
0
250
350
2300
AMP
400
ATP
0
300 0
400
2100 2200
500
ADP
400
300
GTP
0
250 0
350 0
400
350 2250 2400
2100
GMP GDP
TABLE I.
600
CDP
0
200 0
300
350 2100
2200
CMP
0
350
2200
500
CTP
0
200
350
2100
0
300
1100
1400
UMP U D P
~
0
300
2250
550
UTP
0
200
350
2150
3' 5' cyclic AMP
0
250
300
2100
a - ~ CH~ATP
0
250
300
2100
~-y CH~ATP
100
r-aceeptor 0
200
300
2200
Without nuelootide
0
250
350
2300
AMP
0
700
1700
500
ADP
GMP
0
750
180
0
700
CMP
0
900
0
200
350
300 2200
1850 1850
400
GDP G T P
0
700
0
750
0
200
350
0
450
900
1400
UMP U D P
500 2100
CTP
1600 1750
600
CDP
0
800
1800
550
UTP
0
200
350
2150
3' 5'eyelieAMP
~-~
0
250
300
2100
CH~ATP
~-T
0
250
300
2100
CH~ATP
p-y
0
200
350
2150
Ctt~UTP
[ 1 4 C ] m a n n o l i p i d . C o n d i t i o n s w e r e as d e s c r i b e d u n d e r t a b l e I. B u t , i n t h i s case, t h e
0
250
350
400 2100
ATP
I n f l u e n c e of di- a n d t r i p h o s p h a t e n u c l e o s i d e s o n e n d o g e n o u s b u f f e r of t h e s e c o n d i n c u b a t i o n c o n t a i n e d Mn ++.
0
200
S - a c e e p t o r CHC13-CH:~OHH~O(1:1:0.3) phase
G D P -[14C] m a n n o s e w a t e r soluble p h a s e
2500
M a n n o l i p i d CHCI3-CH:~OH (2:1) phase
To
TABLE I I.
0
20u
350
2150
~3- y CH~UTP
I n f l u e n c e of di- a n d t r i p h o s p h a t e n u c l e o s i d e s o n e n d o g e n o u s [ 1 4 C ] m a n n o l i p i d . A r e a c t i o n m i x t u r e c o n t a i n i n g G D P - [ 1 4 C ] m a n n o s e a n d lV~n++ w a s i n c u b a t e d a t 30°C f o r 7 m i n u t e s a n d c e n t r i f u g e d . T h e p e l l e t c o n t a i n i n g l a b e l e d e n d o g e n o u s l i p i d w a s s u b s e q u e n t l y w a s h e d f o u r t i m e s w i t h 0.0~5 M T r i s - m a l e a t e p H ~ 6, 0.25 M~ s u c r o s e to r e m o v e l a b e l e d G D P - m a n n o s e , m a n n o s e - p h o s p h a t e a n d ~ n +÷. T h e w a s h e d p a r t i c l e s w e r e t h e n r e s u s p e n d e d i n t h e w a s h i n g b u f f e r a n d i n c u b a t e d w i t h 1 m M n u c l e o t i d e a t 30°C f o r 10 m i n u t e s . A t t h e e n d of t h i s s e c o n d i n c u b a t i o n p e r i o d , t h e r e a c t i o n m i x t u r e w a s e x t r a c t e d as d e s c r i b e d i n M a t e r i a l a n d M e t h o d s . (To : w i t h o u t s e c o n d i n c u b a t i o n ) . R e s u l t s a r e e x p r e s s e d i n c p m .
G D P -[i4C] m a n n o s e w a t e r soluble p h a s e
100
200
"~ S ,. a c c e p t o r CHC13-CH3OHH20(l:l:0.3) phase
r- acceptor
2500
To
M a n n o l i p i d CHCIa-CH3OH (2:1) phase
~V
~°
.°
f~
7-
M. Richard, F. Tytgat and P. Louisot.
598
tion. F o r this p u r p o s e , nuclei w e r e d e l i p i d a t e d [203 and i n c u b a t e d w i t h p u r i f i e d [14C] m a n n o l i p i d , w i t h or w i t h o u t guanine-nncleotides, w i t h or w i t h o u t Mn 2+. T h e s e e x p e r i m e n t s do not m o d i f y the p r e v i o u s conclusion.
Kinetics of [l~C~mannose incorporation from S-acceptor. As s h o w n in figure 5, w h e n i s o l a t e d [14C]man6 °)
nose-labeled S - a c c e p t o r is used as substrate, r a d i o a c t i v i t y d i s a p p e a r s from S - a c c e p t o r w i t h the c o n c o m i t a n t a p p e a r a n c e of r a d i o a c t i v i t y in r - a c c e p t o r and in the aqueous p h a s e (X comp o u n d ) of the f r a c t i o n a t e d i n c u b a t i o n m i x t u r e . M~n2+ w a s a n e c e s s a r y metal ion.
o
E o=
~
= E
o
j~,mannolipid
/J 15
30
S i m i l a r results, b u t less conclusive, w e r e observed w h e n s o d i u m d e o x y c h o l a t e w a s r e p l a c e d b y T r i t o n X-100. 7 °) Properties of ~annosyl S-acceptor. A c i d h y d r o l y s i s of [ l ~ C ] m a n n o s y l S - a c c e p t o r r e l e a s e d o n l y free [14C]mannose. A l k a l i n e h y d r o lysis w i t h 10 p e r cent NH4OH for 2 h o u r s at 100°C r e l e a s e d a w a t e r - s o l u b l e p r o d u c t that h a d i d e n t i cal p r o p e r t i e s w i t h X c o m p o u n d . By gel-filtration on S e p h a d e x G-5@, a p p a r e n t m o l e c u l a r w e i g h t w a s a p p r o x i m a t e l y 3,000. The r e l a t i v e m i g r a t i o n s (R F) w i t h s i l i c a gel in systems A, B, C, D, E, w e r e r e s p e c t i v e l y 0.06, 0, 0.13, 0.47 60, n ° 6-7.
8 °) Properties of X compound. As s h o w n in figure 5, a large p a r t of the r a d i o a c t i v i t y a p p e a r s in the w a t e r - s o l u b l e f r a c t i o n of the i n c u b a t i o n m i x t u r e . The aqueous phase cont a i n e d a single r a d i o a c t i v e c o m p o u n d m i g r a t i n g , w h e n s u b j e c t e d to h i g h voltage e l e c t r o p h o r e s i s , as an anion in p y r i d i n buffer as well as in b o r a t e buffer. By t r e a t m e n t w i t h alkaline p h o s p h a t a s e , it was c o n v e r t e d to a n e u t r a l c o m p o u n d in p y r i d i n buffer. Acid h y d r o l y s i s and a - m a n n o s i d a s e incub a t i o n s l i b e r a t e d all of the r a d i o a c t i v i t y as free mannose. Dy gel filtration analysis, X c o m p o u n d e x h i b i t e d a m o l e c u l a r w e i g h t of 1,3~0-1,400. 9) Characterization of mannosyl-r-acceptor. Solubilization of the insoluble r e s i d u e cont a i n i n g m a n n o s y l - r - a c c e p t o r w i t h 0.5 p e r cent s o d i u m d e o x y c h o l a t e , f o l l o w e d b y gel filtration on S e p h a d e x G-50 i n d i c a t e d that l a b e l e d m a n n o s e was l i n k e d to a c o m p o n e n t w h i c h h a d a m o l e c u l a r w e i g h t of 36,000. P r o n a s e - t r e a t e d m a n n o s y l - r a c c e p t o r s w e r e low m o l e c u l a r w e i g h t f r a g m e n t s as a d j u d g e d b y gel filtration a n d t r i c h l o r o a c e t i c a c i d solubility. Strong a c i d h y d r o l y s i s of l a b e l e d m a n n o s y l - r - a c c e p t o r l i b e r a t e d all of the r a d i o a c t i vity as free m a n n o s e .
°° = 60
minutes Fro. 5. - - [14C]mannose transfer from S-aeceptor to endogenous protein. The incubation mixtures were fractionated as described in Material and l~ethods.
BIOCHIMIE, 1 9 7 8 ,
and 0:5~. A n i s a l d e h y d d e t e c t i o n located a greenish spot c o n t a i n i n g all the a p p l i e d r a d i o a c t i v i t y .
Discussion.
Among studied rat liver nuclear glycosyltransferases, only the m a n n o s y l t r a n s f e r a s e was able to t r a n s f e r sugar on the c o m p o n e n t s e x t r a c t e d in the lipid-solvents. I n d e e d , u n l i k e m i c r o s o m i c systems, t h e r e was no [14C]N-acetyl-glucosamine t r a n s f e r f r o m UDP-N-acetyl-[z4C]g l u c o s a m i n e into the CHC13-CH30H (2:1) extracts ( u n p u b l i s h e d results). T h e k i n e t i c s of [14CJmannose i n c o r p o r a t i o n from GD'P-[z4C.]mannose s h o w a p r e c u r s o r p r o duct relationship between S-acceptor and r-accept o t a n d are c o n s i s t e n t w i t h those o b t a i n e d in different non n u c l e a r m e m b r a n o u s systems [7, 13]. P u r i f i e d [14C]mannolipid h a d p r o p e r t i e s of a m a n n o s y l - p h o s p h o r y l - d o l i c h o l (Dol-P=man) : lability to dilute acid, s t a b i l i t y to alkaline t r e a t m e n t and, above all, t h i n - l a y e r c h r o m a t o g r a p h i c identity to a u t h e n t i c Dol-P-[14C]Man. W h e n exogenous [14CJmannolipid was tested as a m a n n o s e donor,
Nuclear mannosyl-transferase
o n l y m a n n o s y l - S - a c c e p t o r w a s l a b e l e d . U n l i k e diff e r e n t r e s u l t s [13, 21, 22], t h e r e w a s no l a b e l - t r a n s fer i n t o r - a c c e p t o r . R e a c t i o n m i s c a r r i a g e m a y be d u e to 0.,5 p e r c e n t T r i t o n X-100, u s e d to a d d m a n n o l i p i d into i n c u b a t i o n m i x t u r e . Incubation nuclear fraction with exogenous [ 1 4 C ] m a n n o s e - l a b e l e d S - a c c e p t o r l e a d e d to a l a b e l incorporation into insoluble (r-acceptor) and w a t e r - s o l u b l e (X c o m p o u n d ) phases. Mn z+ s e e m e d to be n e c e s s a r y to t h i s t r a n s f e r r e a c t i o n . A l k a l i n e treatment of p u r i f i e d [~4C]mannose labeled S - a c c e p t o r r e s u l t e d in c o n v e r s i o n of the r a d i o a c t i v i t y to a w a t e r - s o l u b l e f o r m . T h i s m a t e r i a l w a s i d e n t i c a l to t h e X c o m p o u n d : it m i g r a t e d as an a n i o n w h e n s u b j e c t e d to p a p e r e l e c t r o p h o r e s i s ; it w a s c o n v e r t e d to a n e u t r a l c o m p o u n d b y alkaline p h o s p h a t a s e t r e a t m e n t ; a c i d h y d r o l y s i s a n d a - m a n n o s i d a s e l i b e r a t e d all of t h e r a d i o a c t i v i t y as f r e e m a n n o s e ; it e x h i b i t e d a m o l e c u l a r w e i g h t of 1,300-1,400. On t h e basis of t h e s e data, w e h a v e c o n c l u d e d t h a t t h e X c o m p o u n d w a s a [14C]mannose-containing oligosaccharide phosphate. T h e X c o m p o u n d a p p a r e a n c e in t h e a q u e o u s s o l u b l e f r a c t i o n r e s u l t e d of d e g r a d a t i o n of S - a c c e p tor w h i c h h a d b e e n i n t r o d u c e d in the i n c u b a t i o n m i x t u r e . T h e q u e s t i o n is to say if the X c o m p o u n d r e s u l t e d f r o m an e n z y m a t i c effect or a d i r e c t a n d s p e c i f i c a c t i o n of d e o x y c h o l a t e . T h e first h y p o thesis m a y n o t be e l i m i n a t e d a c t u a l l y . F o r t h e second, quite similar results had been obtained by Hsu [22] on m y e l o m a m i c r o s o m e p r e p a r a t i o n w i t h T r i t o n X-100. A d e t e r g e n t effect is p o s s i b l e w h i c h m a y f a v o u r an e n z y m a t i c h y d r o l y s i s b u t an e v e n t u a l s p e c i f i c i t y d e o x y c h o l a t e m a y be eliminated. A l k a l i n e t r e a t m e n t of S - a c c e p t o r and t h e o b s e r v a t i o n t h a t this c o u m p o u n d is m o r e a c i d i c t h a n m a n n o s y l - p h o s p h o r y l - d o l i c h o l (S~acceptor D E A E c e l l u l o s e c o l u m n w a s e l u t e d w i t h 0.1 M a m m o n i u m a c e t a t e i n s t e a d of 0.02 M for Dol.P-Man.) s u g g e s t e d t h a t t h e o l i g o s a c c h a r i d e w a s l i n k e d to t h e l i p i d m o i e t y by a p y r o p h o s p h a t e b o n d , as d e s c r i b e d by l~ehrens et al. ~23]. T h e l i p i d m o i e t y w a s d e t e c t e d by a n i s a l d e h y d r e a c t i o n . I~4C]mannose r-acceptors were glycoproteins : after pronase treatment, the products were low m o l e c u l a r w e i g h t f r a g m e n t s as a d j u g e d b y gel filtration and trichloroacetic acid solubility. D i f f e r e n t n u c l e o t i d e s w e r e t e s t e d on t h e e n d o g e n o u s [ l ~ C ] m a n n o l i p i d . W i t h di- a n d t r i - p h o s p h a t e s n u c l e o s i d e s , an i n c r e a s e of t h e r a d i o a c t i v i t y w a s o b s e r v e d in t h e o l i g o s a c c h a r i d e - l i p i d a n d g l y c o p r o t e i n s . T h e s e r e s u l t s are not in a g r e e m e n t
BIOCHIMIE, 1978, 60, n ° 6-7.
: lipids intermediates.
599
w i t h t h o s e r e p o r t e d b y d i f f e r e n t a u t h o r s [7, 13] b u t m a y be c o m p a r e d w i t h t h o s e o b t a i n e d in t h y r o i d [24]. In c o n c l u s i o n , m a n n o l i p i d s are i n t e r m e d i a t e s in m a n n o s e t r a n s f e r to r a t l i v e r n u c l e i g l y c o p r o reins ; h o w e v e r n u c l e a r m a n n o s y l - t r a n s f e r a s e m e c h a n i s m does n o t c o r r e s p o n d q u i t e to L e n n a r z ' s scheme [~].
Acknowledgements. We are indebeted to Dr. F. W. Hemming (Liverpool) for his generous gift of [l~C]mannosyl-phosphoryldolichol. This work was supported by the ¢ Centre National de la Recherche Scientifique (ERA 562), the ¢ l n s t i l n t Nalional de la Sant~ et de la Recherche Mddicale>), the c Ddldgation Gdndrale h la Recherche Scientifique et Technique ~>, the c Direction des Recherehes et Moyens d'Essais >>, the <(Fonclation pour la Recherche Mddieale 7> and the University of Lyon.
REFERI~NCES. 1. Riehards, J. B. a Hemming, F. W. (1972) Bioehem. J., 130, 77-9'3. 2. Behrens, ~. H., Carminati, H., Staneloni, R. J., Leloir, L. F. a Cantarella, A. I. (1973) Proc. Nat. Aead. Sci. U.S.A., 70, 3390-3394. 3. Maestri, N. & De Luea, L. (1973) Biochem. Biophys. Res. Commun., 53~ 1344-1349. 4. Baynes, J. W., H,su, A. F. & Heath, E. C. (197'3) J. Biol., Chem., 248, 569,3-5704. 5. Babczinski, P. & Tanner, W. (1973) Bioehem~ Biophys. Res. Commun., 54, 1119~1124. 6. Letoublou, R. & Got, R. (1974) FEBS Letters, 46, 214247. 7. Chambers, J. & Elhein, A. D. (I9,76) J. Biol. Chem., 250, 6904-6915. 8. Rogge, H. & Risse, H. J. (1973) Hoppe Seyler's Z. Physiol. Chem., ~54, 1236. 9. Rogge, H. & Risse, H. J. (1.974) Hoppe Seyler's Z. Physiol. Chem., 3~55, 1244-1245. 10. Richard, M., Martin, A. & Louisot, P. (1975) Biochem. Biophys. Res. Commun., 64, 1'08-114. 11. Hubert, M. T., Favard, P., Carasso, N.,.Rosenewajg, R. & Zalta, J. P. (1962) J. Microscopze, 1, 435-444. 12. Beck, J. P., Guerne, J. M., Schmitt, G., S~tutinsky, F. & Ebel, J. P. (1968) ~C. R. Acad. Sci., 266, 940-943. 13. Waechter, C. J., Lucas, J. J. & Lennarz, W. J. (1973) J. Biol. Chem., 248, 7570'-7579. 14. Lester, R. L. & Steiner, M. R. (19~8) J. Biol. Chem., 243, 4889-4893. 15. Mc Sweeney, G. P. (1965) J. Chromatogr., 17, 183185. 16. Wawszkiewiez, E. J. (1961) Analytic. Chem., 33, 252'-254. 17. Folch, J., Lees, M. ~ Sloane-Stanley, G. H. (1957) J. Biol. Chem., 226, 49~7-50918. Zilversmit, D. B. (1960) Am. J. Med., 29, 832-848. 19. Dallner, G., Behrens, N. H., Parodi, A. J. & Leloir, L. F. (1972) FEBS Letters, 24, 315-317. 20. Troy, F. A., Frerman, F. E. ~ Heath, E. C. (1971) J. Biol. Chem., 246, 118-133. 21. Waechter, C. J., Lueas, J. J. ~ Lennarz, W. J. (1974) Biochem. Biophys. Res. Commun., 56, 343-350. 22. Hsu An-Fei, Bayn~s, J. W. & Heath, E. C. (1974) Proe. Nat. Acad. Set. U.S.A., 71, 2391-2395. 23. Behrens, N. H., Parodi, A. J. a Leloir, L. F. (1971) Proc. Nat. Acad. Sci. U.S.A., 68, 2~857-2860. 24. Ronin, C. & Bouchilloux, S. (19.76) Biochim. Biophys. Acta, 428, 445-455. 25. Lennarz, W. J. (1975). Science, 188, 986-991.
BIOCHIMIE, 1978, 60, 5'9'3-599.
Study of nuclear mannosyl-transferase : lipids intermediates. M. R I C H A R D (*), F. T Y T G A T a n d P. L O U I S O T ~.
University o[ Lyon, Lyon-Sud Medical School, Laboratory o[ B i o c h e m i s t r y and ERA-CNBS, 562, 69600 Oullins, France.
R6sum6.
Summary.
La m m m o s y l - t r a n s f 6 r a s e de n o y a u de foie de rat c a t a l y s e le transfert du m a n n o s e , & partir du G D P - m a n n o s e , sur des a c c e p t e u r s e n d o q ~ n e s lipidiques o u prot6iniques. Deux q l y c o l i p i d e s d e solubilit6s diff6rentes ont 6t6 caract6ris6s : un m a n n o s y l - p h o s p h o r y l - d o l i c h o l et un oliqosaccharide-lipide. Le m a r q u a q e radioactif de ces deux lipides est c o m p a t i b l e a v e c le r61e qu'ils p e u v e n t jouer d a n s le transfert du m a n n o s e a u x prot6ines nucl6aires, m a i s p a s s e l o n le m6can i s m e de Lennarz.
M a n n o s y l - t r a n s f e r a s e of rat liver nuclei catal y z e d the transfer of m a n n o s e , from GDP-mann o s e , to e n d o q e n o u s lipids a n d proteins. T w o solubility-different qlycolipids w e r e characterized : a mannosyl-phosphoryl-dolichol and an oliqosaccharide-lipid. The labelinq of the two lipids w a s consistent with a role in m a n n o s e transfer to n u c l e a r qlycoproteins, but not accordinq to the Lennarz' s c h e m e .
Introduction.
Material and Methods.
I n t h e l a s t t h r e e y e a r s , m a n y w o r k s [1-7] h a v e shown the part taken by polyprenic compounds i n t h e b i n d i n g of o l i g o s a c c h a r i d e c h a i n s t o g l y c o proteins. These works study chiefly the incorpor a t i o n of m a n n o s e a n d N - a c e t y l - g l u c o s a m i n e b y membrane-bound enzymatic systems. P r e v i o u s p a p e r s [8-10] r e p o r t e d g l y c o s y l - t r a n s f e r a s e l o c a l i z a t i o n i n a n u c l e a r cell f r a c t i o n . I n t h e r a t l i v e r n u c l e i [10] e v i d e n c e w a s g i v e n f o r t h e e x i s t e n c e of f i v e g l y c o s y l - t r a n s f e r a s e s : m a n n o s y l - , g a l a c t o s y l - , N - a c e t y l - g l u c o s a m i n y l - , Na c e t y l - g a l a c t o s a m i n y l - a n d s i a l y l - t r a n s f e r a s e . So it s e e m s i n t e r e s t i n g to s t u d y t h e p l a c e of p o l y p r e n i c compounds in nuclear glycoprotein biosynthesis. This paper reports data obtained in such a study, c o n c e r n i n g m a n n o s e b i n d i n g to o l i g o s a c c h a r i d e c h a i n of g l y c o p r o t e i n s .
( * ) Altach6 de R e c h e r c h e s au CNRS.
To whom all correspondence should be addressed.
Key words : mannosyl-transferase, nuclei, glycoproteins, glycolipids, glycosyl-transfera~e.
- - Nuclear enzyme preparation. Rat liver nuclei were prepared according to the Zalta m e t h o d [11] a d a p t e d by Beck el al. [12I. The p u r e n e s s of the nuclear f r a c t i o n was checked by elect r o n microscopy and m a r k e r enzymes [10].
- - A s s a y for incorporation of [l~C]mannose from GDP-[l~C]mannose into endogenous aceepfors. The s t a n d a r d assay m i x t u r e to s t u d y the incorporat i o n of [14C]mannose from GDP-[14C]mannose into the t h r e e endogenous acceptors c o n t a i n e d 0.2' ml of nuclear suspension (1 mg of protein) in 0.05 M Trism a l e a t e buffer pH ~ 6,, 0.25 M sucrose, 0.01 ml of 0.11 M MnCl~ and 0.01 ml of a solution c o n t a i n i n g 117 picomoles of G*DP-[14C]mannose (specific activity 170 Ci/M, NEN Chemicals USA). The r e a c t i o n was started by the a d d i t i o n of GDP-E14C]mannose and carried out at 30°C. The used e x t r a c t i o n procedure was described by W a e c h t e r et al. [13]. The r e a c t i o n was stopped at the indicated t i m e b y the a d d i t i o n of 4.4 ml of CHCI~ - CH3OH (2:1). The m i x t u r e was allowed to s t a n d at room t e m p e r a t u r e for 15' m i n u t e s and t h e n centrifuged. The s u p e r n a t e was removed and the pellet was extracted in the same m a n n e r . The c o m b i n e d CH,CI.~ - - CH,~OH : extracts were t h e n w a s h e d w i t h 2.5 ml of 0.9 per cent NaC1. The upper phase was discarded a n d the lower phase w a s h e d w i t h 3 ml of 0.9 per cent NaCl - - CH, OH (1:0.5). The dried pellet from the CHCI~ ~ CHa0H
594
M. Richard, F. Tytgat and P. Louisot.
e x t r a c t i o n w a s w a s h e d t h r e e t i m e s w i t h 2 m l o f H20 ; the washes were discarded. The pellet was then extract e d t w i c e in 5 m l o f CHCI~ - - CH~OH - - H ~ (1:1:0.3). The residue was conserved. The assay method separated the three [14C]mannose labeled endogenous acceptors : mannolipid, which was s o l u b l e i n C ~ C l z - C H ~ O H (2:1) ; m a n n o s y l - S - a c e e p t o r w h i c h w a s s o l u b l e i n C~CIs - CHzOH, - H~O (1:1:0.3) ; m a n n o s y l - r - a c c e p t o r w h i c h w a s i n s o l u b l e in b o t h of t h e a b o v e s o l v e n t s a n d i n w a t e r ( a c c o r d i n g to L e n n a r z ' s n o m e n c l a t u r e [13]). - - P a r t i a l p u r i f i c a t i o n of [~.',C]mannolipid. T h e o r g a n i c p h a s e f r o m t h e CI~Clz-CHzOH (2:1) e x t r a c t i o n w a s e v a p o r a t e d to d r y n e s s u n d e r r e d u c e d p r e s s u r e . T h e c r u d e l i p i d w a s r e d i s s o l v e d i n CH'CI~CH~O~ (7:3)~ a n d a p p l i e d to a D E A E - c e l l u l o s c c o l u m n (3 X 28 cm) t h a t h a d b e e n e q u i l i b r a t e d i n CHClaCH~OH (7:3). T h e c o l u m n w a s s e q u e n t i a l l y e l u t e d w i t h 10'00 m l of CHCI3 - CH,OIt ( 7 : 3 ) a n d 500 m l of CH~OH. A s m a l l p e r c e n t a g e of t h e r a d i o a c t i v i t y ( a p p r o x i m a t e l y 1.5 p e r c e n t ) w a s r e c o v e r e d i n t h e s e f r a c t i o n s . T h e c o l u m n w a s t h e n e l u t e d w i t h 0.q2 ~ a m m o n i u m acet a t e i n 99 p e r c e n t CH~OH. ~5 p e r c e n t of t h e r a d i o a c t i vity was recovered in a single peak. The fractions cont a i n i n g r a d i o a c t i v i t y w e r e p o o l e d a n d a d d e d to 2 v o l u m e s of CHCI~. T h e m i x t u r e w a s w a s h e d w i t h 1/5 v o l u m e of H20 to r e m o v e a m m o n i u m a c e t a t e . T h e o r g a n i c p h a s e w a s e v a p o r a t e d to d r y n e s s u n d e r r e d u c e d p r e s s u r e a n d d i s s o l v e d i n 3 m l of CI-I~OH - t o l u e n e (1:1) a n d t r e a t e d w i t h a l k a l i [14]. T h e d e a c y l a t e d l i p i d f r a c t i o n ( a p p r o x i m a t e l y 80 p e r c e n t of t h e r a d i o a c t i v i t y ) w a s a p p l i e d to a n o t h e r DEAE-cellulose column (2 X 15 era) e q u i l i b r a t e d in CI-IEI~- CH~OH (7~:3). T h e c o l u m n w a s e l u t e d w i t h 109 m l o f GH,CI~- CH~OH. (7:3), 100 m l o f CH:~OH a n d a c o n t i n u o u s g r a d i e n t of a m m o n i u m a c e t a t e (0-0:03 M9 i n m e t h a n o l . 7~ p e r c e n t of t h e r a d i o a c t i v i t y w e r e e l u t e d in a s i n g l e s y m m e t r i c a l peak with the continuous gradient. Ammonium acetate w a s r e m o v e d as p r e v i o u s l y d e s c r i b e d . T h e l i p i d w a s s t o r e d a t - - 20°C. - - P a r t i a l p u r i f i c a t i o n of m a n n o s y l - S - a c c e p t o r . T h e C~C13 - CH3OH - H~O (1:1:0.3) extracts were a p p l i e d to a DEA~E-ccllulose C o l u m n (3 X 25 c m ) , t h a t h a d b e e n e q u i l i b r a t e d i n CHCI~ - CH~OH - H~O (1:1:0.3). T h e c o l u m n w a s w a s h e d w i t h 500 m l of CHCla - CHaOH H~O (1:1:0.3), (no r a d i o a c t i v i t y w a s r e c o v e r e d in t h i s f r a c t i o n ) , t h e n c l u t e d w i t h 0.1 M a m m o n i u m a c e t a t e i n CHCI~- CH~Otf- H~O (1:1:0:3). I n t e r m e d i a t e s t e p s w i t h l o w e r s a l t c o n c e n t r a t i o n s e l u t e d 5 p e r c e n t of t h e radioactivity. The labeled fractions were pooled, conc e n t r a t e d , w a s h e d to r e m o v e a m m o n i u m a c e t a t e a n d s t o r e d at - - 2 0 ° C . -- Chromatographic procedures. Partially purified mannolipid was thin-layer chrom a t o g r a p h e d o n s i l i c a gel. T h e d e v e l o p i n g s o l v e n t s w e r e : A, C~CI~ - CH~OH - CH~COOH - H~O (26~:16:14:2) ; B, C H C I ~ - C H ~ O H - N H ~ O H (75:25:4) : C. n - p r o p a n o l H~O (8:2). T h e p o l y p r e n o l s w e r e d e t e c t e d b y e x p o s i n g t h e c h r o m a t o g r a m s to a n i s a l d e h y d [15]. l ~ a n n o s y l - S - a c c e p t o r w a s c h r o m a t o g r a p h e d o n silica gel w i t h s o l v e n t s A, B, C, D, n - p r o p a n o l - H~O'(7:3) a n d E, n - p r . o p a n o l - H~O (5:3). GDP-mannose, mannose-phosphate and mannose w e r e s e p a r a t e d o n W h a t m a n N ° 3 MM: p a p e r in t h e s o l v e n t F , e t h a n o l - M CH~COO N : a p H ; = 3 . 6 (75:30), B ! O C H I M I E , 1978, 60, .u ° 6-7.:
mannose-l-phosphate and mannose-6-phosphate on E c t e o l a - - c e l l u l o s e i n t h e s o l v e n t G, 95 p e r c e n t e t h a nol --0.1 M a m m o n i u m t e t r a b o r a t e pIff = 9 (3:2% These compounds were detected by exposing the chrom a t o g r a m s to p e r i o d a t e - b e n z i d i n c [16]. N e u t r a l s u g a r s w e r e c h r o m a t o g r a p h e d o n s i l i c a gel i n t h e s o l v e n t H, l ' - b u t a n o l - p y r i d i n e - 0 . 1 N HE1 (5:3:2). S t a n d a r d s u g a r s w e r e l o c a t e d b y a n i l i n e phtalate. -- Hydrolytic procedures. C o n d i t i o n s f o r acid h y d r o l y s i s of m a n n o l i p i d a r e described in the figure legend. Alkaline hydrolysis was p e r f o r m e d b y 0.1 N N a O H in 90 p e r c e n t e t h a n o l at 80°C f o r 15 m i n u t e s . D i f f e r e n t c o m p o u n d s w e r e e x t r a c t e d b y t h e F o l c h ' s m e t h o d [17]. W a t e r - s o l u b l e p r o d u c t s w e r e e h r o m a t o g r a p h e d o n ,l~cteola-ccllulose i n t h e solv e n t G. M a n n o s y l - S - a e c e p t o r s , p r e p a r e d f r o m GI)P-[14C] m a n n o s e or [ 1 4 C ] m a n n o l i p i d , m a n n o s y l - r - a e c e p t o r s a n d X c o m p o u n d s (see r e s u l t s ) w e r e t r e a t e d b y 3N HC1 at l(r0°C f o r 3 h o u r s . H y d r o l y s a t e s w e r e f r a c t i o n a t e d on D o w e x 51) X 4 (200-400 m e s h ) a n d D o w e x 1 × 8 (200400 m e s h ) . C o n c e n t r a t e d e l u a t e s w e r e c h r o m a t o g r a p h e d o n silica gel i n t h e s o l v e n t H. -- Enzyme digestion. Mannosyl-r-aeeeptor prepared from GDP-mannose a n d m a n n o s y l - s - a c e e p t o r w a s i n c u b a t e d i n 5 m l of 0.05 M Tris-H)C1 p ~ = 8, 0.01 m l of M, CaCl~, 1 m g of p r o n a s e . A d r o p of t o l u c n w a s a d d e d a n d t h e r e a c t i o n w a s c a r r i e d o u t a t 37°C. A d d i t i o n a l p r o n a s c (0.5 rag) w a s a d d e d a t 5 a n d 24 h o u r s . At 48 h o u r s t h e m i x t u r e w a s f r o z e n f o r l a t e r use. X c o m p o u n d (see r e s u l t s ) w a s i n c u b a t e d w i t h a l k a l i n e p h o s p h a t a s e ( W o r t h i n g t o n ] b i o c h e m i c a l Corp.) i n 0.1 M Tris-HC1, p H 7.8, a t 37°C f o r 3 h o u r s or w i t h a - m a n n o s i d a s e ( B o e h r i n g e r ) in 0.5 M s o d i u m c i t r a t e , p H = 4.5 at 37°C f o r 36 h o u r s . - - Gel f i l t r a t i o n . A S e p h a d e x G-50 c o l u m n (2.5)< 50 em), w a s p r e p a r e d a n d e q u i l i b r a t e d w i t h 0.06 M Tris-HC1, p H : 7.2, c o n t a i n i n g 0.5 p e r c e n t s o d i u m d e o x y e h o l a t e . T h e c o l u m n was calibrated with proteins, baeitraein and sucrose. U n t r e a t e d or p r o n a s e - t r e a t e d m a n n o s y l - r - a e e e p t o r s , m a n n o s y l - s - a e e e p t o r a n d X - c o m p o u n d w e r e a p p l i e d to the column. -- Paper electrophoresis. X c o m p o u n d s (see r e s u l t s ) w e r e a p p l i e d to W h a t m a n No 3 MM p a p e r a n d s u b j e c t e d to e l e e t r o p h o r e s i s (40 v o l t s p e r e m f o r 1.5 h o u r s ) w i t h p y r i d i n e - CH~COOHH~O (pH : 6.5) or 1 p e r c e n t s o d i u m t e t r a b o r a t e , p H = 9.5. - - A s s a y f o r i n c o r p o r a t i o n of [ l ' , C ] m a n n o l i p i d a n d m a n n o s y l - S - a c c e p t o r into e n d o g e n o u s acceptors. P a r t i a l l y p u r i f i e d m a u n o l i p i d , e v a p o r a t e d to d r y n e s s u n d e r r e d u c e d p r e s s u r e , w a s d i s s o l v e d in a 5 p e r c e n t T r i t o n X-l,00 s o l u t i o n (in t h i s d e t e r g e n t t h e d i s . s o l u t i o n w a s c o m p l e t e ) . 0.02 m l w e r e a d d e d to 0.2 m l of n u c l e a r s u s p e n s i o n (1-1.5 m g p r o t e i n ) w i t h or w i t h o u t Mn2+. F i n a l c o n c e n t r a t i o n of T r i t o n X-100 w a s 0.5 p e r cent. D i f f e r e n t c o m p o u n d s w e r e e x t r a c t e d as d e s c r i b e d a b o v e . M a u n o s y l - S - a e c e p t o r w a s i n t r o d u c e d in t h e s y s t e m b y s u b s t i t u t i o n of T r i t o n X-100 w i t h 5 p e r c e n t s o d i u m
Nuclear mannosyl-transferase deoxycholate. method.
Products
were
extracted
by
: lipids intermediates.
59.5
usual v
--- Routine methods. P r o t e i n w a s d e t e r m i n e d b y the m e t h o d of Lowry. Radioactivity was m e a s u r e d on a P a c k a r d Tri-Carb liquid s c i n t i l l a t i o n counter.
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1 °) K i n e t i c s o[ [ l ~ C ] m a n n o s e f r o m GDP-[1~C]mannose.
V
incorporation
T h e t i m e c o u r s e of i n c o r p o r a t i o n of m a n n o s e from GDP-[~4C]mannose into three endogenous a c c e p t o r s i n t h e n u c l e a r f r a c t i o n is s h o w n i n E
I0
3.
,~ 2i 3~
3
2
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S GDP- Man.
F]6. 2. - - 2a : melal ion requirement for mannolipid synthesis. 2b : dependence on pH of mannolipid synthesis. 2 e : dependence on temperature of mannolipid synthesis. 2d : dependence on GDP-mannose concentration of mannolipid synthesis.
i' 'o
1~
3'0
~'o
minutes FIG. 1 . Time course of [l$C]mannose transfer from GDP-[I~C] mannose into rat liver nuclei. Mannolipid, S-aeeeptor a n d r-aeeeptor were obtained as deseribed in Material a n d Methods.
3"
"~2
"K o e-
f i g u r e 1. T h e s e t h r e e p r o d u c t s w e r e d e f i n e d b y their solubility properties. The mannolipid is s o l u b l e i n CHC13 -CaX-IaOH (2:1), m a n n o s y l - S - a c c e p t o r is s o l u b l e i n CHC13 - CH3OH - H 2 0 (1,:1:0.3) a n d m a n n o s y l - r - a c c e p t o r is f o u n d i n t h e i n s o l u b l e p e l let. It is e v i d e n t f r o m figure 1 t h a t l a b e l e d m a n n o l i p i d is f o r m e d at a m u c h g r e a t e r r a t e ( × 10) a n d at h i g h e r l e v e l s t h a n m a n n o s y l - S - a c c e p t o r a n d , after 7 minutes, undergoes turnover. Mannosyl-ra c c e p t o r is f o r m e d m o r e s l o w l y a n d is m e t a b o l i c a l l y s t a b l e . D e s i d e s , it is n o t e w o r t h y t h a t e x i s t s BIOCHIMIE, 1978, 60, n ° 6-7.
El
'
2'0
4'0
'
6'0 minutes
Fro. 3. - - Mild acid hydrolysis of mannolipid. The reaction was carried out at 35°C in 2.5 ml of 0.1 N H,CI in CH~OH. At the indicated times, 0.1 ml was w i t h d r a w n a n d t r a n s f e r r e d to a m i x t u r e of 2 ml of CHCI.~CH~Ott (2:1) a n d 0.5 ml of 0.1)2 N NaOH - - The m i x t u r e was mixed a n d the r a d i o a c t i v i t y was m e a s u r e d in the aqueous a n d organic phases.
M. Richard, F. T y t g a t and P. Louisot.
596
a p r e c u r s o r - d e r i v a t i v e relation a c c o r d i n g to Zilv e r s m i t [183 b e t w e e n S-acceptor and r-acceptor. 2 °) Conditions [or synthesis of mannolipid.
It was i n t e r e s t i n g to verify the reversibility of this enzymatic reaction. E x p e r i m e n t s were carried out on a n u c l e a r s u s p e n s i o n c o n t a i n i n g preformed, endogenous [14CJmannolipid w i t h diffe-
The t r a n s f e r of [l~C]mannose from C~D'P-[z4C] m a n n o s e into m a n n o l i p i d (fig 2a) requires a divalent metal ion, Mn 2÷ being nlore effective t h a n Mg 2÷. The optimal c o n c e n t r a t i o n of Mn 2÷ is approximately 0.005 M. The optimal pH for m a n n o l i p i d synthesis is about 6 (fig. 2b), the optimal temperature about 30°C (fig. 2c)'. F r o m the d e p e n d e n c e of the f o r m a t i o n of labeled m a n n o l i p i d on GD~P-mannose c o n c e n t r a t i o n s h o w n in figure 2d, an appar e n t K M for GDP-mannose of 0.6 t~M is calculated. 3 °) Properties of partially purified mannolipid. a) T r e a t m e n t of m a n n o l i p i d w i t h alkali a n d acid. T r e a t m e n t of the p a r t i a l l y p u r i f i e d m a n n o l i p i d w i t h 0.1 N HC1 at 35°C resulted in a r a p i d release of free mannose. The k i n e t i c of h y d r o l y s i s s h o w n in figure 3 indicates that the t 1/2 for the m a n n o lipid is 15 minutes. Mannose was the only p r o d u c t formed. The m a n n o l i p i d was stable to m i l d alkaline methanolysis (0.1 N NaOH, at 35°C for 60 minutes). A water-soluble p r o d u c t was released by strong alkaline h y d r o l y s i s (0.1 N NaOH in 90 per cent ethanol at 80°C for 16 minutes). The water-soluble product had a c h r o m a t o g r a p h i c m o b i l i t y s i m i l a r to that of m a u n o s e - l - p h o s p h a t e on Ecteola-cellulose in solvent system 6. b) C h r o m a t o g r a p h i c properties of m a n n o l i p i d . T h i n - l a y e r c h r o m a t o g r a p h i e s of partially purified [ l ~ C ] m a n n o l i p i d , on silica gel, in solvents A, B, C, exhibited R r, respectively 0.Y5, 0.40 and 0.78. W i t h these three solvent mixtures, [~*C]mannol i p i d was c h r o m a t o g r a p h i c a l l y identical to a u t h e n tic [a4C]mannosyl-phosphoryl-dolichol p r e p a r e d by Dr. Hemming.
4 °) Mannolipid as a precursor of mannosyl-Sacceptor. Exogenous p a r t i a l l y p u r i f i e d [14C]mannolipid was tested as a m a n n o s e donor. The results s h o w n in figure ~ reveal that n u c l e a r fraction catalyzed the t r a n s f e r of [14C]mannose from exogenous m a n n o l i p i d into mannosyl-S-acceptor. No watersoluble p r o d u c t and no r-acceptor was detected. This r e a c t i o n did not r e q u i r e Mn 2+. 5 °) Effects o[ nucleotides on endogenous man-
nolipid. M a n n o l i p i d is synthesized a c c o r d i n g to the reaction s h o w n below : L i p i d -4- ~ D P - m a u n o s e Mn2+ m a n n o l i p i d + GDP
BIOCHIMIE, 1978, 60, n ° 6-7.
-
1.5
t,.
,--
t~ £2
.s
tO
= /
~.racceptor,aqueous phase. 15
FIG. 4.
exogenous
-
-
30
fi'O
minutes. Synthesis of mannosyl S-acceptor from [l~C]mannolipid. Incubation conditions
were described in Material and Methods.
rent nucleotides, w i t h or w i t h o u t Mn 2~. GDP-[14C] m a n n o s e absence of r e i n c u b a t i o n m e d i u m was verified by high voltage electrophoresis. F r o m the tables I and H, it appears : a d i m i n u t i o n of the r a d i o a c t i v i t y i n the CHC13 -CH3OH (2:1) extracts w i t h di-and triphosphate nucleosides. Monophosphate-nucleosides, 3'-5' cyclic AMP and n o n h y d r o l y s a b l e nucleotides had no effect. - no GDP-[l~C]mannose at the end of the reinc u b a t i o n period. - -
- - an increase of the r a d i o a c t i v i t y in the CHC13 CHaOH- H20 (1:1:0.3) extracts with di-and triphosphate-nueleosides. - - an increase of the r a d i o a c t i v i t y in the r-aceeptor w i t h d i - a n d t r i p h o s p h a t e - n u c l e o s i d e s and Mn 2+. I n view of these results that are not in favour of the r e v e r s i b i l i t y of m a n n o l i p i d synthesis, we have used a different method. Results o b t a i n e d by D a l l n e r et al [19] have s h o w n that nuclei have a very high c o n c e n t r a t i o n of dolichol-phosphate, that w o u l d p r e v e n t the r e v e r s i b i l i t y of t h e reac-
0
0
200
300
2200
Without nucleotide
0
250
350
2300
AMP
400
ATP
0
300 0
400
2100 2200
500
ADP
400
300
GTP
0
250 0
350 0
400
350 2250 2400
2100
GMP GDP
TABLE I.
600
CDP
0
200 0
300
350 2100
2200
CMP
0
350
2200
500
CTP
0
200
350
2100
0
300
1100
1400
UMP U D P
~
0
300
2250
550
UTP
0
200
350
2150
3' 5' cyclic AMP
0
250
300
2100
a - ~ CH~ATP
0
250
300
2100
~-y CH~ATP
100
r-aceeptor 0
200
300
2200
Without nuelootide
0
250
350
2300
AMP
0
700
1700
500
ADP
GMP
0
750
180
0
700
CMP
0
900
0
200
350
300 2200
1850 1850
400
GDP G T P
0
700
0
750
0
200
350
0
450
900
1400
UMP U D P
500 2100
CTP
1600 1750
600
CDP
0
800
1800
550
UTP
0
200
350
2150
3' 5'eyelieAMP
~-~
0
250
300
2100
CH~ATP
~-T
0
250
300
2100
CH~ATP
p-y
0
200
350
2150
Ctt~UTP
[ 1 4 C ] m a n n o l i p i d . C o n d i t i o n s w e r e as d e s c r i b e d u n d e r t a b l e I. B u t , i n t h i s case, t h e
0
250
350
400 2100
ATP
I n f l u e n c e of di- a n d t r i p h o s p h a t e n u c l e o s i d e s o n e n d o g e n o u s b u f f e r of t h e s e c o n d i n c u b a t i o n c o n t a i n e d Mn ++.
0
200
S - a c e e p t o r CHC13-CH:~OHH~O(1:1:0.3) phase
G D P -[14C] m a n n o s e w a t e r soluble p h a s e
2500
M a n n o l i p i d CHCI3-CH:~OH (2:1) phase
To
TABLE I I.
0
20u
350
2150
~3- y CH~UTP
I n f l u e n c e of di- a n d t r i p h o s p h a t e n u c l e o s i d e s o n e n d o g e n o u s [ 1 4 C ] m a n n o l i p i d . A r e a c t i o n m i x t u r e c o n t a i n i n g G D P - [ 1 4 C ] m a n n o s e a n d lV~n++ w a s i n c u b a t e d a t 30°C f o r 7 m i n u t e s a n d c e n t r i f u g e d . T h e p e l l e t c o n t a i n i n g l a b e l e d e n d o g e n o u s l i p i d w a s s u b s e q u e n t l y w a s h e d f o u r t i m e s w i t h 0.0~5 M T r i s - m a l e a t e p H ~ 6, 0.25 M~ s u c r o s e to r e m o v e l a b e l e d G D P - m a n n o s e , m a n n o s e - p h o s p h a t e a n d ~ n +÷. T h e w a s h e d p a r t i c l e s w e r e t h e n r e s u s p e n d e d i n t h e w a s h i n g b u f f e r a n d i n c u b a t e d w i t h 1 m M n u c l e o t i d e a t 30°C f o r 10 m i n u t e s . A t t h e e n d of t h i s s e c o n d i n c u b a t i o n p e r i o d , t h e r e a c t i o n m i x t u r e w a s e x t r a c t e d as d e s c r i b e d i n M a t e r i a l a n d M e t h o d s . (To : w i t h o u t s e c o n d i n c u b a t i o n ) . R e s u l t s a r e e x p r e s s e d i n c p m .
G D P -[i4C] m a n n o s e w a t e r soluble p h a s e
100
200
"~ S ,. a c c e p t o r CHC13-CH3OHH20(l:l:0.3) phase
r- acceptor
2500
To
M a n n o l i p i d CHCIa-CH3OH (2:1) phase
~V
~°
.°
f~
7-
M. Richard, F. Tytgat and P. Louisot.
598
tion. F o r this p u r p o s e , nuclei w e r e d e l i p i d a t e d [203 and i n c u b a t e d w i t h p u r i f i e d [14C] m a n n o l i p i d , w i t h or w i t h o u t guanine-nncleotides, w i t h or w i t h o u t Mn 2+. T h e s e e x p e r i m e n t s do not m o d i f y the p r e v i o u s conclusion.
Kinetics of [l~C~mannose incorporation from S-acceptor. As s h o w n in figure 5, w h e n i s o l a t e d [14C]man6 °)
nose-labeled S - a c c e p t o r is used as substrate, r a d i o a c t i v i t y d i s a p p e a r s from S - a c c e p t o r w i t h the c o n c o m i t a n t a p p e a r a n c e of r a d i o a c t i v i t y in r - a c c e p t o r and in the aqueous p h a s e (X comp o u n d ) of the f r a c t i o n a t e d i n c u b a t i o n m i x t u r e . M~n2+ w a s a n e c e s s a r y metal ion.
o
E o=
~
= E
o
j~,mannolipid
/J 15
30
S i m i l a r results, b u t less conclusive, w e r e observed w h e n s o d i u m d e o x y c h o l a t e w a s r e p l a c e d b y T r i t o n X-100. 7 °) Properties of ~annosyl S-acceptor. A c i d h y d r o l y s i s of [ l ~ C ] m a n n o s y l S - a c c e p t o r r e l e a s e d o n l y free [14C]mannose. A l k a l i n e h y d r o lysis w i t h 10 p e r cent NH4OH for 2 h o u r s at 100°C r e l e a s e d a w a t e r - s o l u b l e p r o d u c t that h a d i d e n t i cal p r o p e r t i e s w i t h X c o m p o u n d . By gel-filtration on S e p h a d e x G-5@, a p p a r e n t m o l e c u l a r w e i g h t w a s a p p r o x i m a t e l y 3,000. The r e l a t i v e m i g r a t i o n s (R F) w i t h s i l i c a gel in systems A, B, C, D, E, w e r e r e s p e c t i v e l y 0.06, 0, 0.13, 0.47 60, n ° 6-7.
8 °) Properties of X compound. As s h o w n in figure 5, a large p a r t of the r a d i o a c t i v i t y a p p e a r s in the w a t e r - s o l u b l e f r a c t i o n of the i n c u b a t i o n m i x t u r e . The aqueous phase cont a i n e d a single r a d i o a c t i v e c o m p o u n d m i g r a t i n g , w h e n s u b j e c t e d to h i g h voltage e l e c t r o p h o r e s i s , as an anion in p y r i d i n buffer as well as in b o r a t e buffer. By t r e a t m e n t w i t h alkaline p h o s p h a t a s e , it was c o n v e r t e d to a n e u t r a l c o m p o u n d in p y r i d i n buffer. Acid h y d r o l y s i s and a - m a n n o s i d a s e incub a t i o n s l i b e r a t e d all of the r a d i o a c t i v i t y as free mannose. Dy gel filtration analysis, X c o m p o u n d e x h i b i t e d a m o l e c u l a r w e i g h t of 1,3~0-1,400. 9) Characterization of mannosyl-r-acceptor. Solubilization of the insoluble r e s i d u e cont a i n i n g m a n n o s y l - r - a c c e p t o r w i t h 0.5 p e r cent s o d i u m d e o x y c h o l a t e , f o l l o w e d b y gel filtration on S e p h a d e x G-50 i n d i c a t e d that l a b e l e d m a n n o s e was l i n k e d to a c o m p o n e n t w h i c h h a d a m o l e c u l a r w e i g h t of 36,000. P r o n a s e - t r e a t e d m a n n o s y l - r a c c e p t o r s w e r e low m o l e c u l a r w e i g h t f r a g m e n t s as a d j u d g e d b y gel filtration a n d t r i c h l o r o a c e t i c a c i d solubility. Strong a c i d h y d r o l y s i s of l a b e l e d m a n n o s y l - r - a c c e p t o r l i b e r a t e d all of the r a d i o a c t i vity as free m a n n o s e .
°° = 60
minutes Fro. 5. - - [14C]mannose transfer from S-aeceptor to endogenous protein. The incubation mixtures were fractionated as described in Material and l~ethods.
BIOCHIMIE, 1 9 7 8 ,
and 0:5~. A n i s a l d e h y d d e t e c t i o n located a greenish spot c o n t a i n i n g all the a p p l i e d r a d i o a c t i v i t y .
Discussion.
Among studied rat liver nuclear glycosyltransferases, only the m a n n o s y l t r a n s f e r a s e was able to t r a n s f e r sugar on the c o m p o n e n t s e x t r a c t e d in the lipid-solvents. I n d e e d , u n l i k e m i c r o s o m i c systems, t h e r e was no [14C]N-acetyl-glucosamine t r a n s f e r f r o m UDP-N-acetyl-[z4C]g l u c o s a m i n e into the CHC13-CH30H (2:1) extracts ( u n p u b l i s h e d results). T h e k i n e t i c s of [14CJmannose i n c o r p o r a t i o n from GD'P-[z4C.]mannose s h o w a p r e c u r s o r p r o duct relationship between S-acceptor and r-accept o t a n d are c o n s i s t e n t w i t h those o b t a i n e d in different non n u c l e a r m e m b r a n o u s systems [7, 13]. P u r i f i e d [14C]mannolipid h a d p r o p e r t i e s of a m a n n o s y l - p h o s p h o r y l - d o l i c h o l (Dol-P=man) : lability to dilute acid, s t a b i l i t y to alkaline t r e a t m e n t and, above all, t h i n - l a y e r c h r o m a t o g r a p h i c identity to a u t h e n t i c Dol-P-[14C]Man. W h e n exogenous [14CJmannolipid was tested as a m a n n o s e donor,
Nuclear mannosyl-transferase
o n l y m a n n o s y l - S - a c c e p t o r w a s l a b e l e d . U n l i k e diff e r e n t r e s u l t s [13, 21, 22], t h e r e w a s no l a b e l - t r a n s fer i n t o r - a c c e p t o r . R e a c t i o n m i s c a r r i a g e m a y be d u e to 0.,5 p e r c e n t T r i t o n X-100, u s e d to a d d m a n n o l i p i d into i n c u b a t i o n m i x t u r e . Incubation nuclear fraction with exogenous [ 1 4 C ] m a n n o s e - l a b e l e d S - a c c e p t o r l e a d e d to a l a b e l incorporation into insoluble (r-acceptor) and w a t e r - s o l u b l e (X c o m p o u n d ) phases. Mn z+ s e e m e d to be n e c e s s a r y to t h i s t r a n s f e r r e a c t i o n . A l k a l i n e treatment of p u r i f i e d [~4C]mannose labeled S - a c c e p t o r r e s u l t e d in c o n v e r s i o n of the r a d i o a c t i v i t y to a w a t e r - s o l u b l e f o r m . T h i s m a t e r i a l w a s i d e n t i c a l to t h e X c o m p o u n d : it m i g r a t e d as an a n i o n w h e n s u b j e c t e d to p a p e r e l e c t r o p h o r e s i s ; it w a s c o n v e r t e d to a n e u t r a l c o m p o u n d b y alkaline p h o s p h a t a s e t r e a t m e n t ; a c i d h y d r o l y s i s a n d a - m a n n o s i d a s e l i b e r a t e d all of t h e r a d i o a c t i v i t y as f r e e m a n n o s e ; it e x h i b i t e d a m o l e c u l a r w e i g h t of 1,300-1,400. On t h e basis of t h e s e data, w e h a v e c o n c l u d e d t h a t t h e X c o m p o u n d w a s a [14C]mannose-containing oligosaccharide phosphate. T h e X c o m p o u n d a p p a r e a n c e in t h e a q u e o u s s o l u b l e f r a c t i o n r e s u l t e d of d e g r a d a t i o n of S - a c c e p tor w h i c h h a d b e e n i n t r o d u c e d in the i n c u b a t i o n m i x t u r e . T h e q u e s t i o n is to say if the X c o m p o u n d r e s u l t e d f r o m an e n z y m a t i c effect or a d i r e c t a n d s p e c i f i c a c t i o n of d e o x y c h o l a t e . T h e first h y p o thesis m a y n o t be e l i m i n a t e d a c t u a l l y . F o r t h e second, quite similar results had been obtained by Hsu [22] on m y e l o m a m i c r o s o m e p r e p a r a t i o n w i t h T r i t o n X-100. A d e t e r g e n t effect is p o s s i b l e w h i c h m a y f a v o u r an e n z y m a t i c h y d r o l y s i s b u t an e v e n t u a l s p e c i f i c i t y d e o x y c h o l a t e m a y be eliminated. A l k a l i n e t r e a t m e n t of S - a c c e p t o r and t h e o b s e r v a t i o n t h a t this c o u m p o u n d is m o r e a c i d i c t h a n m a n n o s y l - p h o s p h o r y l - d o l i c h o l (S~acceptor D E A E c e l l u l o s e c o l u m n w a s e l u t e d w i t h 0.1 M a m m o n i u m a c e t a t e i n s t e a d of 0.02 M for Dol.P-Man.) s u g g e s t e d t h a t t h e o l i g o s a c c h a r i d e w a s l i n k e d to t h e l i p i d m o i e t y by a p y r o p h o s p h a t e b o n d , as d e s c r i b e d by l~ehrens et al. ~23]. T h e l i p i d m o i e t y w a s d e t e c t e d by a n i s a l d e h y d r e a c t i o n . I~4C]mannose r-acceptors were glycoproteins : after pronase treatment, the products were low m o l e c u l a r w e i g h t f r a g m e n t s as a d j u g e d b y gel filtration and trichloroacetic acid solubility. D i f f e r e n t n u c l e o t i d e s w e r e t e s t e d on t h e e n d o g e n o u s [ l ~ C ] m a n n o l i p i d . W i t h di- a n d t r i - p h o s p h a t e s n u c l e o s i d e s , an i n c r e a s e of t h e r a d i o a c t i v i t y w a s o b s e r v e d in t h e o l i g o s a c c h a r i d e - l i p i d a n d g l y c o p r o t e i n s . T h e s e r e s u l t s are not in a g r e e m e n t
BIOCHIMIE, 1978, 60, n ° 6-7.
: lipids intermediates.
599
w i t h t h o s e r e p o r t e d b y d i f f e r e n t a u t h o r s [7, 13] b u t m a y be c o m p a r e d w i t h t h o s e o b t a i n e d in t h y r o i d [24]. In c o n c l u s i o n , m a n n o l i p i d s are i n t e r m e d i a t e s in m a n n o s e t r a n s f e r to r a t l i v e r n u c l e i g l y c o p r o reins ; h o w e v e r n u c l e a r m a n n o s y l - t r a n s f e r a s e m e c h a n i s m does n o t c o r r e s p o n d q u i t e to L e n n a r z ' s scheme [~].
Acknowledgements. We are indebeted to Dr. F. W. Hemming (Liverpool) for his generous gift of [l~C]mannosyl-phosphoryldolichol. This work was supported by the ¢ Centre National de la Recherche Scientifique (ERA 562), the ¢ l n s t i l n t Nalional de la Sant~ et de la Recherche Mddicale>), the c Ddldgation Gdndrale h la Recherche Scientifique et Technique ~>, the c Direction des Recherehes et Moyens d'Essais >>, the <(Fonclation pour la Recherche Mddieale 7> and the University of Lyon.
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