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 1993, 175(7):2102.J. Bacteriol.

V Mrsa, F Klebl and W Tanner a cell wall endo-beta-1,3-glucanase.

product,Saccharomyces cerevisiae BGL2 genePurification and characterization of the

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JOURNAL OF B A C T E R I O L O G Y , A p r . 1 9 9 3 , p . 2 1 0 2 - 2 1 0 60 0 2 1 - 9 1 9 3 / 9 3 / 0 7 2 1 0 2 - 0 5 0 2 . 0 0 / 0C o p y r i g h t © 1 9 9 3 , A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y

V o l . 1 7 5 , N o . 7

P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f t h e S a c c h a r o m y c e sc e r e v i s i a e BGL2 G e n e P r o d u c t , a C e l l

W a l l E n d o - j 3 - 1 , 3 - G l u c a n a s eVLADIMIR MRSA,t F R A N Z KLEBL, A N D WIDMAR T A N N E R *

L e h r s t u h l f i r Z e l i b i o l o g i e , U n i v e r s i t d t R e g e n s b u r g , U n i v e r s i t d t s s t r a s s e 3 1 , 8 4 0 0 R e g e n s b u r g , G e r m a n y

R e c e i v e d 1 4 D e c e m b e r 1 9 9 2 / A c c e p t e d 1 F e b r u a r y 1 9 9 3

O n e o f t h e m a j o r p r o t e i n s o f t h e S a c c h a r o m y c e s c e r e v i s i a e c e l l w a l l , a 1 3 - g l u c a n a s e ( B G L 2 g e n e p r o d u c t ) , h a sb e e n i s o l a t e d a n d p u r i f i e d t o h o m o g e n e i t y u n d e r c o n d i t i o n s f o r p r e s e r v i n g e n z y m e a c t i v i t y . T h e s t u d y o fe n z y m e p r o p e r t i e s o f t h e p r o t e i n r e v e a l e d t h a t i t i s a n e n d o - 0 - 1 , 3 - g l u c a n a s e a n d n o t a n e x o g l u c a n a s e a s

r e p o r t e d p r e v i o u s l y ( F . K l e b l a n d W. T a n n e r , J . B a c t e r i o l . 1 7 1 : 6 2 5 9 - 6 2 6 4 , 1 9 8 9 ) . T h e e x am i na t i o n o f t h eg l u c a n a s e s t r u c t u r e s h o w e d t h a t t h e l o w e r a p p a r e n t m o l e c u l a r m a s s o f t h e p r o t e i n ( 2 9 k D a ) c o m p a r e d w i t hw h a t w a s c a l c u l a t e d f r o m t h e a m i n o a c i d s e q u e n c e o f t h e e n z y m e ( 3 3 . 5 k D a ) i s d u e t o a n o m a l o u s m i g r a t i o n i ns o d i u m d o d e c y l s u l f a t e g e l s a n d n o t t o p o s t t r a n s l a t i o n a l p r o c e s s i n g o f t h e p o l y p e p t i d e c h a i n . Of t w o p o t e n t i a lN g l y c o s y l a t i o n s i t e s a t A s n - 2 0 2 a n d A s n - 2 8 4 , o n l y t h e l a t t e r s i t e i s g l y c o s y l a t e d . T h e o v e r p r o d u c t i o n o f t h e1 3 - g l u c a n a s e f r o m t h e h i g h - c o p y - n u m b e r p l a s m i d b r o u g h t a b o u t a s i g n i f i c a n t d e c r e a s e i n t h e g r o w t h r a t e o ft r a n s f o r m e d y e a s t c e l l s .

W i t h r e g a r d t o t h e v a r i o u s g l u c a n a s e a c t i v i t i e s d e s c r i b e d

f o r S a c c h a r o m y c e s c e r e v i s i a e , t w o g e n e s , BGL1 a n d B G L 2 ,h a v e b e e n c l o n e d   5 , 6 , 1 1 ) . B o t h c o r r e s p o n d i n g g e n e p r o d -u c t s a r e t r a n s p o r t e d t h r o u g h t h e c e l l m e m b r a n e , w i t h t h eBGLJ p r o d u c t b e i n g m a i n l y s e c r e t e d i n t o t h e m e d i u m ( 1 1 )w h i l e t h e BGL2 p r o d u c t i s i n c o r p o r a t e d i n t o t h e c e l l w a l l   5 .T h e l a c k o f e i t h e r p r o t e i n d i d n o t r e s u l t i n a p h e n o t y p e   5 ,1 1 ) . T h e BGLI g e n e h a s b e e n i d e n t i f i e d b y s e l e c t i n g f o ro v e r p r o d u c e r s   6 , a n d i t s e e m s t o c o d e f o r t h e m a i n e x o - p -g l u c a n a s e a c t i v i t y o f y e a s t c e l l s ( 1 1 ) . T h e BGL2 g e n e s h o w e da s i g n i f i c a n t s e q u e n c e s i m i l a r i t y t o p l a n t j 3 - g l u c a n a s e s   5 .T h e BGL2 g e n e p r o d u c t was o r i g i n a l l y p u r i f i e d a s a d o m i -n a n t c e l l w a l l p r o t e i n w i t h a n a p p a r e n t m o l e c u l a r mass o f 2 9

k D a . A l t h o u g h t h e p u r i f i c a t i o n i n c l u d e d a h e a t s t e p , t h ep u r i f i e d p r o t e i n s t i l l s h o w e d m i n o r 1 3 - 1 , 3 - g l u c a n a s e a c t i v i t y  5 . To s t u d y t h i s p r o t e i n i n more d e t a i l , i t wa s p u r i f i e d t o

h o m o g e n e i t y u n d e r c o n d i t i o n s f o r p r e s e r v i n g e n z y m e a c t i v -i t y . T h e e n z y m e i s n o t , a s p r e v i o u s l y d e s c r i b e d   5 , a ne x o g l u c a n a s e b u t a n e n d o - , 3 - 1 , 3 - g l u c a n a s e . P h y s i c o c h e m i c a la n d e n z y m a t i c p r o p e r t i e s o f t h e p r o t e i n h a v e b e e n s t u d i e d .

MATERILS A N D METHODS

T h e f o l l o w i n g s t r a i n s were u s e d : S . c e r e v i s i a e X - 2 1 8 0 - 1 A MATa SUC2 m a l g a l 2 C U P J ) a n d S E Y 2 1 0 1 ( M A T a u r a 3 - 5 2l e u 2 - 3 , 1 1 2 a d e 2 - 1 s u c 2 - A 9 g a l 2 )   3 a n d E s c h e r i c h i a c o l iD H ~ a [ F - e n d i L h s d R l 7 ( r K - m K j ) s u p E 4 4 t h i - 1 r e c A lg y r A 6 9 r e l A l 4 8 0 d l a c Z A M 1 5 J ( 4 ) .

C h e m i c a l s . [ 1 4 C ] l a m i n a r i n wa s k i n d l y p r o v i d e d b y H .

K a u s s , K a i s e r s l a u t e r n , G e r m a n y . A l l o t h e r c h e m i c a l s w e r eo f t h e h i g h e s t p u r i t y c o m m e r c i a l l y a v a i l a b l e .

1 3 - G l u c a n a s e p u r i f i c a t i o n . As s h o w n p r e v i o u s l y ( 5 ) , t h eBGL2 g e n e p r o d u c t s p l i t s l a m i n a r i n ; t h e r e f o r e , t h e a m o u n to f r a d i o a c t i v i t y r e l e a s e d f r o m [ 1 4 C ] l a m i n a r i n was u s e d t od e t e r m i n e t h e enzyme a c t i v i t y . I n a d d i t i o n , t h e p u r i f i c a t i o n

* C o r r e s p o n d i n g a u t h o r .

t P r e s e n t a d d r e s s : B i o c h e m i s t r y D e p a r t m e n t , F a c u l t y o f F o o dT ec h n o l o g y a nd B i o t e c h n o l o g y , U n i v e r s i t y o f Z a g r e b , 4 1 0 0 0 Z a -g r e b , C r o a t i a .

was c h e c k e d b y m o n i t o r i n g t h e 2 9 - k D a b a n d b y i m m u n o b l o ta f t e r s e p a r a t i o n o n s o d i u m d o d e c y l s u l f a t e ( S D S ) g e l s . F o re n z y m e p u r i f i c a t i o n , c e l l s o f S . c e r e v i s i a e X - 2 1 8 0 - 1 A w e r eg r o w n i n 1 l i t e r o f YPD medium ( 1 y e a s t e x t r a c t , 1p e p t o n e , 2 D - g l u c o s e ) t o t h e l a t e l o g a r i t h m i c p h a s e , h a r -v e s t e d b y c e n t r i f u g a t i o n , w a s h e d w i t h w a t e r , a n d r e s u s -p e n d e d i n 5 m l o f 2 0 mM s o d i u m p h o s p h a t e b u f f e r , pH 7 . 0 .C e l l s w e r e d i s r u p t e d m e c h a n i c a l l y i n a B r a u n MSK h o m o g -e n i z e r , a n d c e l l d e b r i s w e r e c e n t r i f u g e d a t 2 0 , 0 0 0 x g a n dw a s h e d w i t h b u f f e r . Z y m o l y a s e 1 0 0 T ( S e i k a g a k u Kogyo C o .L t d . , T o k y o , J a p a n ) was a d d e d t o t h e s u s p e n s i o n c o n t a i n i n gc e l l w a l l s a t a c o n c e n t r a t i o n o f 6 j u g / m l , a n d t h e m i x t u r e wasi n c u b a t e d f o r 1 h a t 3 7 0 C . C e l l w a l l s w e r e t h e n c e n t r i f u g e d

a n d t h e s u p e r n a t a n t wa s d i a l y z e d a g a i n s t 1 0 mM s o d i u mp h o s p h a t e b u f f e r , pH 6 . 2 . T h e enzyme p r e p a r a t i o n was

a p p l i e d t o a D E A E - S e p h a r o s e CUB c o l u m n ( 1 . 6 b y 5 c m )a n d e l u t e d b y a l i n e a r 0 t o 4 0 0 mM N a C l g r a d i e n t a t a f l o wr a t e o f 2 0 m l / h . T h r e e - m i l l i l i t e r f r a c t i o n s w e r e c o l l e c t e d a n da n a l y z e d f o r t h e 2 9 - k D a p r o t e i n b y i m m u n o b l o t a nd f o r t h e

p - n i t r o p h e n y l g l u c o s i d a s e a c t i v i t y . T h e a c t i v i t y a g a i n s t p - n i -t r o p h e n y l g l u c o s i d e was f o u n d t o b e c o m p l e t e l y s e p a r a t e df r o m t h e 2 9 - k D a p r o t e i n p e a k ( d a t a n o t s h o w n ) . T h e 2 9 - k D ag l u c a n a s e f r a c t i o n s w e r e c o l l e c t e d a n d a p p l i e d t o a Mono Qh i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ( H P L C ) c o l u m n( H i 5 / 5 ; P h a r m a c i a ) i n 1 0 mM s o d i u m p h o s p h a t e b u f f e r , pH6 . 2 , a n d e l u t e d w i t h a 0 t o 5 0 0 mM N a C l g r a d i e n t a t 1 m l / m i n .T h e p e a k c o n t a i n i n g t h e g l u c a n a s e was a p p l i e d t o a TSK2 0 0 0 HPLC c o l u m n ( U l t r o p a c k c o l u m n TS K G 2 0 0 0 S W ;

LKB) e q u i l i b r a t e d w i t h 2 0 mM s o d i u m p h o s p h a t e b u f f e r , pH6 . 2 , c o n t a i n i n g 0 . 1 M N a C l a n d was e l u t e d w i t h t h e s a m eb u f f e r . F r a c t i o n s c o n t a i n i n g t h e enzyme w e r e p o o l e d a n dc o n c e n t r a t e d b y l y o p h i l i z a t i o n .

1 3 - G l u c a n a s e a c t i v i t y d e t e r m i n a t i o n . T h e e n z y m e a c t i v i t yw a s m e a s u r e d w i t h [ 1 4 C ] l a m i n a r i n a s t h e s u b s t r a t e . T h er e a c t i o n m i x t u r e c o n t a i n e d 3 , u g o f l a m i n a r i n ( s p e c i f i c r a d i o -a c t i v i t y , 1 0 6 c p m / m g ) a n d t h e a p p r o p r i a t e a m o u n t o f enzymei n 2 0 p A o f 5 0 mM s o d i u m a c e t a t e b u f f e r , pH 5 . 5 . S o d i u ma z i d e was a d d e d a s a p r e s e r v a t i v e t o a f i n a l c o nc en t r at i o n o f0 . 1 . T h e r e a c t i o n was c a r r i e d o u t f o r 2 t o 6 0 m i n a t 3 0 0 C ,a n d t h e r e a c t i o n m i x t u r e wa s t h e n a p p l i e d t o t h i n - l a y e r

2 1 0 2

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S . CEREVISIAE BGL2 P-GLUCANASE 2 1 0 3

T A B L E 1 . P u r i f i c a t i o n o f t h e R - g l u c a n a s e

Step A m t o f A c t i v i t y S p a c t

  t p p r o t e i n ( m g )   u m o n l m i n p r m o l / m i n . m g )

Z y m o l y a s e e x t r a c t 3 8 . 0 _aD E A E - S e p h a r o s e CL6B 4 . 3 0 . 3 1 5 0 . 0 7 3

Mono Q 1 . 2 0 . 2 8 3 0 . 2 3 6

TS K 2 0 0 0 0 . 3 0 . 1 4 1 0 . 4 7 0

  A c t i v i t y o f t h e Z y m o l y a s e e x t r a c t was no t i n c l u d e d b e c a u s e i t i s p a r t i a l l yd u e t o o t h e r g l u c a n as e s p r e s e n t i n t h e e x t r a c t . I n f u r t h e r p u r i f i c a t i o n s t e p s ,

o t h e r g l u c a n a s e a c t i v i t i e s were s e p a r a t e d f r o m t h e 2 9 - k D a g l u c a n a s e ( c h e c k e d

w i t h i m m u no b l o t s ) .

c h r o m a t o g r a p h y on c e l l u l o s e p l a t e s ( M e r c k ) . T h e c h r o m a t o -

gram was d e v e l o p e d f o u r t i me s i n a c e t o n e - n - b u t a n o l - w a t e r

( 7 0 : 1 5 : 1 5 ) , a n d t h e r a d i o a c t i v i t y was s c a n n e d .P r o t e i n e s t i m a t i o n was d o n e by t h e m e t h o d o f Lowry e t a l .

( 8 ) .P r e p a r a t i o n o f t h e a n t i - 1 3 - g l u c a n a s e a n t i s e r u m . P o l y c l o n a l

a n t i b o d i e s were o b t a i n e d b y t h e p r o c e d u r e o f B u r n e t t e ( 1 ) . AN e w Z e a l a n d W h i t e r a b b i t was i m m u n i z e d w i t h t h e p u r i f i e d

3 - g l u c a n a s e as f o l l o w s . A p r i m a r y i n j e c t i o n c o n t a i n i n g 8 0 p u g

o f p r o t e i n i n c o m p l e t e F r e u n d ' s a d j u v a n t was f o l l o w e d a f t e r1 4 d a y s b y f o u r b o o s t e r i n j e c t i o n s w i t h 6 0 R g o f p r o t e i n i ni n c o m p l e t e F r e u n d ' s a d j u v a n t a t 8 - d a y i n t e r v a l s . I n a l l c as e s,

t h e a n t i g e n was i n t r o d u c e d b y m u l t i p l e i n t r a d e r m a l i n j e c -t i o n s . B l o o d was t a k e n 2 d a y s a f t e r t h e t h i r d a n d f o u r t h

b o o s t s .

I m m u n o b l o t s . SDS e l e c t r o p h o r e s i s was c a r r i e d o u t b y t h e

m e t h o d o f Laemmli ( 7 ) . F o r i m m u n o b l o t s , g e l s were t r a n s -

f e r r e d t o n i t r o c e l l u l o s e b y t h e m e t h o d o f Dunn ( 2 ) . Then i t r o c e l l u l o s e f i l t e r was i n c u b a t e d f o r 1 . 5 h i n a 1 : 1 , 0 0 0

d i l u t i o n o f a n t i - p - g l u c a n a s e a n t i s e r u m a t room t e m p e r a t u r e .

CNBr p e p t i d e s . P e p t i d e s were p r e p a r e d b y d i g e s t i n g 5 , u go f r a d i o a c t i v e o r 3 0 , u g o f n o n r a d i o a c t i v e 3 - g l u c a n a s e i n 0 . 5m l o f 80 f o r m i c a c i d c o n t a i n i n g 2 5 mg o f CNBr f o r 2 h a t

room t e m p e r a t u r e . P e p t i d e s were t h e n a p p l i e d t o a r e v e r s e d -

p h a s e HPLC column ( H i - P o r e R P 3 0 4 ; 2 5 0 b y 4 . 6 mm;

B i o - R a d ) . P e a k s were e l u t e d w i t h a 0 t o 70 a c e t o n i t r i l eg r a d i e n t a t 1 m l / m i n . R a d i o a c t i v e enzyme was o b t a i n e d b yl a b e l i n g c e l l s w i t h N - [ 1 4 C ] a c e t y l g l u c o s a m i n e , i s o l a t e d , a n d

p u r i f i e d a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . X - 2 1 8 0 - 1 A

c e l l s were  rown i n YPD medium t o a n o p t i c a l d e n s i t y a t 6 0 0

nm o f 2 . 0 , t r a n s f e r r e d t o a medium w i t h o u t g l u c o s e t o w h i c h

1 5 0 , u C i o f N - [ 1 4 C ] a c e t y l g l u c o s a m i n e was a d d e d , a n d i n c u -

1 2 3 4

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F I G . 2 . Time c o u r s e o f [ 4 C ] l a m i n a r i n d e g r a d a t i o n w i t h 3 - g l u c a -n a s e . [ ' 4 C ] l a m i n a r i n was h y d r o l y z e d w i t h 1 3 - g l u c a n a s e as d e s c r i b e d

i n M a t e r i a l s a n d M e t h o d s ; a l i q u o t s o f t h e r ea ct i o n m i x t u r e were

a p p l i e d t o t h i n - l a y e r c h r o m a t o g r a p h y a f t e r 2 ( A ) , 1 0 ( B ) , 3 0 ( C ) , o r

6 0 ( D ) m i n ; a n d t h e s a m p l e s were s c a n n e d f o r r a d i o a c t i v i t y . N u m -b e r s f o r p e a k s a re a s f o l l o w s : 1 , l a m i n a r i n ; 2 , l a m i n a r i t e t r o s e ; 3 ,

l a m i n a r i t r i o s e ; 4 , l a m i n a r i b i o s e ; 5 , g l u c o s e .

b a t e d f o r 1 h a t 3 0 ° C . More t h a n 80 o f t h e r a d i o a c t i v i t y was

i n c o r p o r a t e d .

I n v i t r o t r a n s c r i p t i o n was p e r f o r m e d b y t h e p r o c e d u r e

d e s c r i b e d i n r e f e r e n c e 1 2 .I n v i t r o t r a n s l a t i o n . A r a b b i t r e t i c u l o c y t e l y s a t e t r a n s l a t i o n

k i t  GIBCO-BRL) was u s e d a c c o r d i n g t o t h e d i r e c t i o n s o f

t h e s u p p l i e r a n d o p t i m i z e d f o r M g 2 ' a n d K+ i o n s . R e a c t i o n s

were c a r r i e d o u t w i t h 2 0 0 n g o f RNA i n t h e presence o f 1 5

, u C i o f [ 3 5 S ] m e t h i o n i n e f o r 1 h a t 3 0 ° C . A l l i n c u b a t i o n s were

t e r m i n a t e d by c h i l l i n g t h e m i x t u r e s on i c e a n d were a n a l y z e d

b y S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( P A G E ) .O v e r p r o d u c t i o n o f 1 3 - g l u c a n a s e . C o n s t r u c t i o n s f o r t h e

o v e r p r o d u c t i o n o f P - g l u c a n a s e were p r e p a r e d i n YEp352 b yl i g a t i n g t h e v e c t o r d i g e s t e d w i t h EcoRI a n d a l k a l i n e p h o s -

p h a t a s e w i t h t h e E c o R I - P s t I f r a g m e n t c o n t a i n i n g t h e pro-

moter r e g i o n o f t h e gene a n d t h e P s t I - E c o R I f r a g m e n t

c o n t a i n i n g t h e c o d i n g r e g i o n ( 5 ) . About 5 j i g o f YEp352 a n d

1 0 , g o f e a c h gene f r a g m e n t were u s e d . Th e l i g a t i o n m i x t u r e

was u s e d d i r e c t l y f o r t h e t r a n s f o r m a t i o n o f t h e y e a s t ( s t r a i n

° C

F I G . 3 . Thermal s t a b i l i t y o f P - g l u c a n a s e . Th e p r o t e i n was p r e i n -c u b a t e d a t t h e i n d i c at e d t e m p e r a t u r e s f o r 1 5 m i n .

A C 4

V O L . 1 7 5 , 1 9 9 3

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2 1 0 4 MR9A ET AL.

TAB L E 2 . B i n d i n g o f t h e P - g l u c a n a s e t od i f f e r e n t p o l y s a c c h a r i d e s

P o l y s a c c h a r i d e a d d e d P - G l u c a n a s e d e t e c t e d i n a :

As f i r s t A s s e c o n d P e l l e t S u p e r n a t a n tp a r t n e r p a r t n e r

G l u c a n +

C h i t i n +

L a m i n a r i n G l u c a n - +L a m i n a r i n C h i t i n - +

C h i t o s a n G l u c a n - +

C h i t o s a n C h i t i n - +

G l u c a n L a m i n a r i n +  

G l u c a n C h i t o s a n +  

C h i t i n L a m i n a r i n +  

C h i t i n C h i t o s a n +  

a N o t i c e t h a t c h i t o s a n a n d l a m i n a r i n a r e s o l u b l e i n w a t e r ( s u p e r n a t a n t ) ,w h e r e a s g l u c a n a n d c h i t i n ar e n o t ( p e l l e t ) .

S E Y 2 1 0 1 ) . T r a n s f o r m a n t s were a n a l y z e d f o r o v e r p r o d u c t i o nb y i m m u n o b l o t . O f 4 8 t r a n s f o r m a n t s a n a l y z e d , 6 o v e r p r o -

d u c e r s w e r e f o u n d .

N o r t h e r n ( R N A ) b l o t s w e r e d o n e b y t h e p r o c e d u r e d es c r i b e d b y M a n i a t i s e t a l .   9 .

B i n d i n g t e s t s . P u r i f i e d P - g l u c a n a s e ( 1 0 p u g wa s s h a k e n

w i t h v ar i o u s p o l y s a c c h a r i d e s ( 2 m g / m l ) i n 5 0 mM s o d i u ma c e t a t e b u f f e r , pH 5 . 5 , f o r   5 m i n a t room t e m p e r a t u r e . A f t e r

c e n t r i f u g a t i o n , t h e s u p e r n a t a n t ( 4 0 , u l was e x a m i n e d b ySDS-PAGE a n d s t a i n e d w i t h C o o m a s s i e b l u e   5 , 1 0 ) .

RESULTS A N D DISCUSSION

E n z y m e p u r i f i c a t i o n . R e s u l t s o f a t y p i c a l p u r i f i c a t i o n a r e

p r e s e n t e d i n T a b l e 1 . T h e g l u c a n a s e o b t a i n e d wa s c h e c k e d

f o r p u r i t y b y SDS e l e c t r o p h o r e s i s ( F i g . 1 . T h e p u r i f i c a t i o nd e s c r i b e d r e s u l t e d i n a n enzyme a c t i v i t y w h i c h wa s a t l e a s t1 0 - f o l d h i g h e r t h a n t h a t o b t a i n e d b y t h e m e t h o d i n v o l v i n g t h eh e a t t r e a t m e n t o f c e l l w a l l s   5 .

Enzyme p r o p e r t i e s . A s a l r e a d y n o t i c e d d u r i n g t h e p u r i f i -c a t i o n , t h e 2 9 - k D a p r o t e i n d i d n o t s p l i t t h e s u b s t r a t e p - n i t r o -p h e n y l g l u c o s i d e , w h i c h i s g e n e r a l l y u s e d t o d e t e r m i n e e x o -

, - g l u c a n a s e a c t i v i t y . [ ' 4 C ] l a m i n a r i n , o n t h e o t h e r h a n d , was

r e a d i l y h y d r o l y z e d . T h i n - l a y e r c h r o m a t o g r a p h y o f t h e r e a c -t i o n p r o d u c t s r e v e a l e d f o u r p e a k s c o r r es p o nd i n g t o s a c c h a -r i d e s c o n t a i n i n g o n e t o f o u r g l u c o s e u n i t s ( F i g . 2 ) , s t r o n g l y

i n d i c a t i n g t h a t t h e enzyme i s a n e n d o - , - 1 , 3 - g l u c a n a s e . S u c hr e s u l t s a r e i n c o n t r a s t w i t h t h e f i n d i n g t h a t t h e BGL2 g e n es h o w s s i g n i f i c a n t h o m o l o g i e s t o p l a n t e x o g l u c a n a s e g e n e s ( 5 )a n d t h a t t h e enzyme r e l e a s e s o n l y g l u c o s e i n t h e r e a c t i o nw i t h [ 1 4 C ] l a m i n a r i n ( 5 ) . To r e s o l v e t h i s d i s c r e p a n c y , t h e

c h r o m a t o g r a p h y s y s t e m u s e d i n r e f e r e n c e 5 was t e s t e d w i t hd i f f e r e n t s u g a r s t a n d a r d s a n d f o u n d t o b e i n a p p r o p r i a t e a s i twas n o t a b l e t o d i s t i n g u i s h among d i f f e r e n t p o t e n t i a l l a m i -

n a r i n d e g r a d a t i o n p r o d u c t s ( f o r i n s t a n c e , b e t w e e n g l u c o s ea n d l a m i n a r i b i o s e ) . T h e r e f o r e , i t i s p o s s i b l e t h a t t h e r a d i o -a c t i v e p e a k f o u n d t o c o m i g r a t e w i t h g l u c o s e a c t u a l l y c o n -s i s t e d o f more d e g r a d a t i o n p r o d u c t s . Wh e n c e l l u l o s e p l a t e sw e r e u s e d i n s t e a d o f s i l i c a g e l p l a t e s , a s a l r e a d y m e n t i o n e d ,f o u r p e a k s a p p e a r e d i n t h e c h r o m a t o g r a m .

T h e t i m e c o u r s e o f t h e r e a ct i o n ( F i g . 2 ) s h o w e d t h e d i m e ra n d t r i m e r t o b e m a j o r p r o d u c t s . T h e a m o u n t o f t e t r am er

i n c r e a s e s i n t h e e a r l y p h a s e o f t h e r e a c t i o n , b u t t h i s f o r ms e e m s t o b e f u r t h e r d e g r a d e d t o s m a l l e r p r o d u c t s.

T h e r e a c t i o n s h o w s t h a t t h e o p t i m u m pH i s b e t w e e n 4 . 5a n d 6 . 0 ( d a t a n o t s h o w n ) . S i n c e t h e enzyme was o r i g i n a l l y

f o u n d t o b e a c t i v e a f t e r h e a t t r e a t m e n t o f y e a s t c e l l w a l l s ( 1 0m i n a t 9 5 ° C ) ( 5 ) , i t was c o n s i d e r e d a f a i r l y h e a t - s t a b l ee n z y m e . When t e s t e d f o r h e a t s t a b i l i t y ( F i g . 3 ) , h o w e v e r , i t

d i d n o t s h o w u n u s u a l p r o p e r t i e s i n t h i s r e s p e c t . As a l r e a d y

m e n t i o n e d , t h e p r o t e i n i s o l a t e d p r e v i o u s l y ( 5 ) p o s s e s s e d a tb e s t 5 t o 1 0 o f t h e n a t i v e a c t i v i t y .

I t h a s b e e n s h o w n t h a t t h e 2 9 - k D a 0 - g l u c a n a s e b i n d s t o

c h i t i n i n a d d i t i o n t o g l u c a n a n d l a m i n a r i n ( 5 ) , t h e b i n d i n g s i t e

m o s t p r o b a b l y b e i n g d i f f e r e n t f r o m t h e a c t i v e s i t e o f t h ee n z y m e . I t was a l s o f o u n d t h a t c h i t o s a n , t h e s o l u b l e a n a l o g

o f c h i t i n , c a n b i n d t h e e n z y m e ( d a t a n o t s h o w n ) , a n d t h e

c o m p e t i t i v e e f f e c t s o f t h e s e s u g a r p o l y m e r s w e r e s t u d i e d

( T a b l e 2 ) . I t c o u l d b e c o n c l u d e d t h a t t h e b i n d i n g t o t h e f i r s tp a r t n e r c o u l d p r a c t i c a l l y n o t b e r e v e r s e d b y t h e a d d i t i o n o f

t h e s e c o n d o n e .

I d e n t i f i c a t i o n o f t h e g l y c o s y l a t i o n s i t e . A l t h o u g h t h e p r e -d i c t e d a m i n o a c i d s e q u e n c e o f t h e BGL2 g e n e i n d i c a t e s tw o

p o t e n t i a l g l y c o s y l a t i o n s i t e s , A s n - 2 0 2 a n d A s n - 2 8 4   5 , t h e

s m a l l c h a n g e i n t h e a p p a r e n t m o l e c u l a r mass a f t e r t h e

e n d o g l y c o p e p t i d a s e F t r e a t m e n t f r o m 2 9 t o 2 6 k D a , a n d t h ef a c t t h a t n o i nt e r me di at e b a n d s w e r e d e t e c t a b l e i n l i m i t e dd i g e s t s ( d a t a n o t s h o w n ) , s u g g e s t s t h a t o n l y o n e o l i g o s a c -c h a r i d e c h a i n i s p r e s e n t i n t h e m o l e c u l e . O n t h e o t h e r h a n d ,

t h e m o l e c u l a r mass p r e d i c t e d f r o m t h e DNA s e q u e n c ec o d i n g f o r t h e p r o t e i n m o i e t y i s 3 1 k D a , i . e . , 5 kDa more

t h a n t h e a p p a r e n t m o l e c u l a r mass e s t i m a t e d f r o m S D S -PAGE o f t h e d e g l y c o s y l a t e d p r o t e i n . T h e f o l l o w i n g q u e s -

t i o n s t h e r e f o r e a r i s e . I s t h e BGL2 g e n e p r o d u c t C t e r m i n a l l yp r o c e s s e d b y a n a l o g y t o w h a t h a s b e e n d e s c r i b e d f o r t h et o b a c c o g l u c a n a s e ( 1 3 ) ? I s s u c h a p r o c e s s i n g p o s s i b l y r e -s p o n s i b l e f o r t h e l o s s o f a p o t e n t i a l g l y c o s y l a t i o n s i t e ? To

a n s w e r t h e s e q u e s t i o n s , t h e f o l l o w i n g e x p e r i m e n t s w e r e

c a r r i e d o u t . T h e p r o t e i n wa s s y n t h e s i z e d b y i n v i t r o t r a n -s c r i p t i o n a n d t r a n s l a t i o n . As t h e g l u c a n a s e o b t a i n e d c o u l d

n o t u n d e r g o a n y p o s t t r a n s l a t i o n a l m o d i f i c a t i o n , s u c h a s

s i g n a l s e q u e n c e p r o c e s s i n g , g l y c o s y l a t i o n , o r a t e n t a t i v eC - t e r m i n a l p r o c e s s i n g , t h e m o l e c u l a r mass o f t h e p r o t e i np r e p a r e d i n v i t r o s h o u l d b e i d e n t i c a l t o t h e o n e c a l c u l a t e d ,i . e . , 3 3 . 5 k D a . I n F i g . 4 i t c a n b e s e e n t h a t t h e a p p a r e n t

m o l e c u l a r mass o f 2 9 kDa wa s o b t a i n e d b y SDS e l e c t r o -p h o r e s i s . S u c h a r e s u l t i n d i c a t e s t h a t t h e m o l e c u l a r m a s se s t i m a t e d i s d u e t o t h e a n o m a l o u s m i g r a t i o n o f , B - g l u c a n a s ei n SDS g e l s r a t h e r t h a n t o t h e p r o c e s s i n g o f t h e p o l y p e p t i d ec h ai n o f t h e e n z y m e .

To f i n d o u t w h i c h o f t h e t w o p o t e n t i a l N g l y c o s y l a t i o ns i t e s i s u s e d , t h e p r o t e i n was r a d i o l a b e l l e d i n v i v o w i t h

N - [ 1 4 C ] a c e t y l g l u c o s a m i n e a n d t h e n p u r i f i e d t o h o m o g e n e i t y ;CNBr p e p t i d e s were p r o d u c e d t h e r e f r o m . S i x p e p t i d e s w e r e

kDa1

94

6 8

42

2 6

1 8

1 4

F I G . 4 . S i z e o f , B - g l u c a n a s e o b t a i n e d b y i n v i v o t r a n s c r i p t i o n a n dt r a n s l a t i o n . L a n e s : 1 , p r o t e i n s t a n d a r d s ; 2 , n a t i v e 3 - g l u c a n a s e ; 3 ,e n z y m e d e g l y c o s y l a t e d b y e n d o g l y c o p e p t i d a s e F ; 4 , e n z y m e o b -t a i n e d b y i n v i t r o t r a n s c r i p t i o n a n d t r a n s l a t i o n .

J . B A C T E R I O L .

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S . C E R E V I S I A E BGL2 1 3 - G L U C A N A S E 2 1 0 5

6

5

N . .

F I G . 5 . R e v e r s e d - p h a s e c h r o m a t o g r a p h y o f CNB r p e p t i d es o f P - g l u c a n a s e . The s o l i d l i n e i n d i c a t e s o p t i c a l d e n s i t y a t 2 2 0 n m ; t h e d a s h e dl i n e i n d i c a t e s r a d i o a c t i v i t y ; a n d t h e d o t t e d l i n e i n d i c a t e s a c e t o n i t r i l e c o n c e n t r a t i o n . I n s e t : s c h e m e o f t h e P - g l u c a n a s e , i n c l u d i n g t h e s i g n a ls e q u e n c e ( s t i p p l e d a r e a ) ; M i n d i c a t e s m e t h i o n i n e r e s i d u e s , v e r t i c a l a r r o w s i n d i c a t e t h e tw o p o s s i b l e g l y c o s y l a t i o n s i t e s , a n d CHO s h o w s t h e

s i t e a t w h i c h t h e c a r b o h y d r a t e c h a i n i s a t t a c h e d . N - t e r m i n a l s e q u e n c e s o f t h e m a j o r ( b o l d f a c e t y p e ) a n d m i n o r r a d i o a c t i v e p e p t i d e s a r e a l s os h o w n .

o b t a i n e d a n d s e p a r a t e d b y r e v e r s e d - p h a s e HPLC ( F i g . 5 ) .O n l y o n e p e p t i d e c o n t a i n e d t h e r a d i o a c t i v i t y , a n d when am i x t u r e o f p ep t i de s was p a s s e d t h r o u g h a s m a l l ( 1 - m l )c o n c a n a v a l i n A - S e p h a r o s e c o l u m n p r i o r t o HPLC, t h e p e a kc o r r e s p o n d i n g t o t h i s p e p t i d e was t h e o n l y o n e m i s s i n g i n t h e

c h r o m a t o g r a m ( d a t a n o t s h o w n ) . When t h e g l y c o s y l a t e dp e p t i d e wa s s e q u e n c e d , i t p r o v e d t o b e t h e t e r m i n a l p e p t i d e

( s e e i n s e t i n F i g . 5 ) c o n t a i n i n g t h e s e c o n d p o t e n t i a l g l y c o s y -l a t i o n s i t e a t A s n - 2 8 4 . T h e a m i n o a c i d s e q u e n c e c o n t a i n e d a

s e c o n d m i n o r s e q u e n c e s t a r t i n g w i t h a m i n o a c i d s a f t e r t h et h i r d m e t h i o n i n e , w h i c h i n d i c a t e s t h a t t h e h y d r o l y s i s a t t h el a s t m e t h i o n i n e i n t h e p o l y p e p t i d e was n o t c o m p l e t e . T h i s

a l s o e x p l a i n s t h e s h o u l d e r i n t h e r a d i o a c t i v i t y o f p e a k 3 . I tm a y b e i n t e r e s t i n g t h a t t h e s e c o n d a r y s t r u c t u r e , h y d r o p h o -b i c i t y , a n d s u r f a c e p r o b a b i l i t y p r e d i c t i o n s l o c a t e t h e g l y c o -s y l a t e d A s n - 2 8 4 w i t h i n a h y d r o p h i l i c P - t u r n w i t h a h i g hs u r f a c e p r o b a b i l i t y , w h e r e a s t h e f i r s t g l y c o s y l a t i o n s i t e ( A s n -2 0 2 ) i s l o ca te d w i t h i n t h e p r e d i c t e d r a t h e r h y d r o p h o b i c

3 - p l e a t e d s h e e t i n a r e g i o n w i t h a v e r y l o w s u r f a c e p r o b a -b i l i t y ( d a t a n o t s h o w n ) .

O v e r p r o d u c t i o n . D i s r u p t i o n o f t h e BGL2 g e n e d i d n o tr e s u l t i n a p h e n o t y p e u n d e r l a b o r a t o r y c o n d i t i o n s , n o r wa s

m a t i n g o r s p o r u l a t i o n a f f e c t e d   5 . To t e s t w h e t h e r o v e r p r o -d u ct i o n o f t h e g l u c a n a s e a f f e c t s g r o w t h , o v e r p r o d u c i n gs t r a i n s were c o n s t r u c t e d b y t r a n s f o r m a t i o n w i t h t h e c o r r e -

s p o n d i n g h i g h - co p y - nu m b e r p l a s m i d ( Y E p 3 5 2 ) c o n t a i n i n gt h e BGL2 g e n e . A l t h o u g h o n l y t r a n s f o r m a n t s w i t h a s l i g h to v e r p r o d u c t i o n o f t w o - t o t h r e e f o l d were o b t a i n e d ( s e ei n s e t s i n F i g . 6 ) , p r o b a b l y b e c a u s e h i g h e r o v e r p r o d u c e r sw e r e n o t v i a b l e , t h e y g r e w c o n s i d e r a b l y more s l o w l y t h a n

c o n t r o l c e l l s ( F i g . 6 ) . T h e r e f o r e , t h e f u n c t i o n ( s ) o f t h e

2 9 - k D a P - g l u c a n a s e i n n u l l m u t a n t s , seems t o b e c o m p e n -

F I G . 6 . G r o w t h r a t e o f t h e t r a n s f o r m a n t o v e r p r o d u c i n g 3 - g l u c a -n a s e . 1 , w i l d t y p e ; 2 , c e l l s t r a n s f o r m e d w i t h a c o n s t r u c t i o n h a v i n gt h e BGL2 g e n e b u t l a c k i n g t h e p r o m o t e r r e g i o n ; 3 , c e l l s t r a n s f o r m e dw i t h t h e c o n s t r u c t i o n c o n t a i n i n g t h e f u l l 3 - g l u c a n a s e g e n e . I n s e t A :

N o r t h e r n b l o t o f RNA f r o m o v e r p r o d u c i n g c e l l s ( l a n e 1 a n d t h e w i l d

t y p e ( l a n e 2 . I n s e t B : i m m u n o b l o t o f P - g l u c a n a s e f r o m w i l d - t y p e( l a n e 1 a n d o v e r p r o d u c i n g c e l l s ( l a n e 2 .

VOL. 1 7 5 , 1 9 9 3

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2 1 0 6 MRSA ET AL.

s a t e d , w h i l e l a r g e r a m o u n t s o f t h e p r o t e i n w i t h i n t h e c e l l a r e

a t l e a s t i n h i b i t o r y f o r g r o w t h .

A C K N O W L E D G M E N T S

H e l p f u l d i s c u s s i o n s w i t h L . L e h l e a r e g r a t e f u l l y a c k n o w l e d g e d .T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m t h e D e u t s c h e F o r s -

c h u n g s g e m e i n s c h a f t ( S F B 4 3 ) .

REFERENCES

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