Vol. 186, No. 1, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 15,1992

Pages 190-198

c D N A S E Q U E N C E A N A L Y S I S O F c P g # : R A T LENS FIBER C E L L BEADED-FILAMENT STRUCTURAL P R O T E I N SHOWS H O M O L O G Y T O C Y T O K E R A T 1 N S

Shigeo Masaki* and Tomomasa

Watanabe

Department of Biochemistry, Institute for Developmental Research, Aichi Prefecture Colony, Kasugai, Aichi 480-03, JAPAN

Received

May

30,

1992

SUMMARY: To s t u d y t h e m o l e c u l a r s t r u c t u r e o f t h e g e n e r e s p o n s i b l e f o r a l e n s f i b e r c e l l b e a d e d - f i l a m e n t s t r u c t u r a l p r o t e i n of 9 ~ k D a (CP9#), w e i s o l a t e d i t s s p e c i f i c c D N A f r o m a r a t l e n s c D N A l i b r a r y by u s e o f a n t i - m o u s e CP9tt antiserum. The expressed fusion protein kept the epitopes specific against a n t i - c h i c k C P 9 7 a s well a s a n t i - m o u s e C P 9 ~ a n t i b o d y , a n d t h e s i z e w a s e s t i m a t e d a s 1 9 0 - 2 0 0 k D a , i n d i c a t i n g t h a t t h e c D N A i n s e r t of t h e c l o n e s e e m e d t o e n c o d e a p o l y p e p t i d e w i t h g 0 - 9 0 k D a in a p p e a r a n c e . N o r t h e r n a n a l y s i s i n d i c a t e d t h a t CP9t; m R N A is e x p r e s s e d o n l y in t h e lens, a n d n o t in t h e b r a i n , skin, h e a r t , k i d n e y , lung, a n d l i v e r , a n d t h e s i z e w a s e s t i m a t e d t o 2 . 1 - 2 . 3 k b . In a l e n s o f i n h e r i t e d m i c r o p h t h a l m i c m o u s e , Elo, a t r a c e a m o u n t o f m R N A w i t h the size closely similar to that of rat mRNA was observed. The entire compiled s e q u e n c e ( ] , g 7 3 b p ) s h o w e d a n o p e n r e a d i n g f r a m e c o v e r i n g t h e s e q u e n c e o f 533 a m i n o a c i d s t o t a l l i n g 5 g , g 5 7 D a . No s e q u e n c e h o m o l o g o u s t o t h e e n t i r e CP9tt w a s f o u n d a m o n g t h e e n t r i e s of a n y n u c l e o t i d e a n d a m i n o a c i d s e q u e n c e d a t a b a s e s ; but with respect to a limited amino acid sequence of N-side region of CP9#, a s i g n i f i c a n t h o m o l o g y w i t h c y t o k e r a t i n s w a s f o u n d . © 1992 Academic Press, Inc.

The differentiation mature

of t h e v e r t e b r a t e

eye lens epithelial cell to a

f i b e r c e l l is a d i s t i n c t i v e m o r p h o l o g i c a l e v e n t t h a t i n c l u d e s loss o f

o r g a n e l l e s a n d c e l l e l o n g a t i o n , a n d it is a c c e p t e d system for the study of the mechanism (1). T h e f i r s t f e a t u r e

as a useful experimental

of gene expression during morphogenesis

of t h e e v e n t is b i o c h e m i c a l l y c h a r a c t e r i z e d

by a c h a n g e

in t h e c o m p o s i t i o n of l e n s c r y s t a l l i n s ; a n d a n i n c r e a s e in a c t i n , d e c r e a s e d s y n t h e s i s of v i m e n t i n , t u b u l i n , a n d t h e a c c u m u l a t i o n

of membrane

intrinsic

p r o t e i n s (MIPs) a r e s i m i l a r l y o b s e r v e d (1). Also r e p o r t e d w a s t h e a p p e a r a n c e of a unique cytoskeletal

structure,

the beaded-chain

f i l a m e n t . It is s e e n o n l y

in t h e e l o n g a t i n g l e n s f i b e r c e l l s (2), a n d i t s s t r u c t u r e contains two lens-specific

proteins, referred

*To w h o m c o r r e s p o n d e n c e

should be addressed.

0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

190

in t h e c h i c k m a i n l y

t o a s C P 9 7 a n d C P ~ 9 (3, tt).

Fax: 0568-88-082%

0 5 6 8 - 9 2 - 1335.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

We r e c e n t l y r e p o r t e d t h e d e f i c i e n c y of a 9 # - k i l o d a l t o n (kDa) p r o t e i n , n a m e d C P 9 # , in t h e n o n - c r y s t a l l i n f r a c t i o n f r o m t h e lens of t h e i n h e r i t e d m i c r o p h t h a l m i c mouse, Elo (5). The a n t i b o d y r a i s e d a g a i n s t 9#kDa p r o t e i n r e a c t e d w i t h t h e c h i c k lens CP97 p r o t e i n as well, and an a n t i b o d y against C P 9 7 (4) also r e a c t e d w i t h t h e p r o t e i n . F r o m t h e s e , we c o n c l u d e d t h a t t h e p r o t e i n c o r r e s p o n d s to t h e c h i c k CP97, and n a m e d it ' m o u s e C P 9 4 ' in k e e p i n g w i t h t h e d e s i g n a t i o n . C h i c k CP97 is b e l i e v e d as a t t a c h m e n t

s i t e s for c r y s t a l l i n s and

t h a t it is f u n c t i o n a l l y i m p o r t a n t in lens f i b e r c e l l e l o n g a t i o n and in keeping its c o n f o r m a t i o n , b e c a u s e t h e y a r e w i d e l y d i s t r i b u t e d t h r o u g h o u t t h e lens f i b e r c e l l s and also c l o s e l y a p p r o a c h t h e plasma m e m b r a n e (#). R e c e n t l y F i t z G e r a l d and C a s s e l m a n (6) showed t h a t t h e c o r r e s p o n d i n g p r o t e i n w i t h t h e s a m e i m m u n o r e a c t i v i t y is w i d e l y d i s t r i b u t e d in t h e lens of v a r i o u s a n i m a l s p e c i e s . As C P 9 g had not b e e n p r e v i o u s l y d e s c r i b e d , we c o n c l u d e d t h a t t h e p r o t e i n belongs to t h e c l a s s of lens b e a d e d - f i l a m e n t

structural proteins,

b e c a u s e a n t i b o d y raised a g a i n s t m o u s e C P 9 # r e a c t e d w i t h p u r i f i e d c h i c k C P 9 7 , and v i c e v e r s a (4, 5). B e c a u s e t h e m o l e c u l a r s t r u c t u r e s and t h e r e g u l a t i o n of t h e g e n e s r e s p o n s i b l e for this class of t h e p r o t e i n s h a v e not b e e n studied, we d e c i d e d to i s o l a t e the s p e c i f i c c D N A e n c o d i n g C P 9 # p r o t e i n in this study.

M A T E R I A L S A N D METHODS

The p r e p a r a t i o n of r a b b i t a n t i s e r u m raised a g a i n s t m o u s e lens f i b e r c e l l 94kDa p r o t e i n ( a n t i - C P 9 # a n t i b o d y ) and t h e p r o c e d u r e s of S D S - P A G E and W e s t e r n blot a n a l y s i s w e r e d e s c r i b e d p r e v i o u s l y (5). The rabbit a n t i - c h i c k C P 9 7 a n t i b o d y was t h e g i f t o f Dr. Maisel (t0. The m e t h o d of Huynh et al. ( P r o t o b l o t ® I m m u n o s c r e e n i n g S y s t e m , P r o m e g a C o r p . , Madison, W1, r e f . 7) was used t o s c r e e n t h e lens c D N A e x p r e s s i o n library in ~gt11 ( C l o n t e c h Lab. Inc., P a l o A l t o , CA) p r e p a r e d f r o m young adult SD r a t . DNA inserts f r o m t h e p o s i t i v e c l o n e s w e r e subcloned i n t o a pUC18 v e c t o r , and t h e r e c o r n b i n a n t s w e r e f u r t h e r t r i m m e d w i t h v a r i o u s r e s t r i c t i o n e n z y m e s , e x o n u c l e a s e Ilk and m u n g bean n u c l e a s e (TOYOBO Corp., Japan) and used for s e q u e n c e d e t e r m i n a t i o n of b o t h c o m p l e m e n t a r y s t r a n d s ( S e q u e n a s e Ver.2.0, USB C o r p . , C l e v e l a n d , OH). DNA o l i g o m e r s n a m e d C P P R I ~ 3 ( s e q u e n c e s a r e shown in Fig. #) w e r e s y n t h e s i z e d by J a p a n B i o s e r v i c e Corp. To o b t a i n t h e 3 ' - e n d of C P 9 # c D N A ( 3 ' - R A C E , r e f . 8), 10~g of r a t lens R N A was added to F i r s t - S t r a n d c D N A R e a c t i o n Mix ( c o n t a i n i n g 13 pmol of Not I-d(T)~8 p r i m e r , P h a r m a c i a LKB B i o t e c h . Inc.), and i n c u b a t e d for 2h at 37~. The p r o d u c t s e p a r a t e d f r o m e x c e s s p r i m e r s was c o n c e n t r a t e d , and a d d e d to t h e p o l y m e r a s e c h a i n r e a c t i o n (PCR) c o c k t a i l c o n t a i n i n g 100 pmol of Not 1-d(T)~ ~ p r i m e r , 100 pmol of C P P R 3 and 2.5 units of A m p l i t a q ® DNA p o l y m e r a s e ( P e r k i n - E l m e r C e t u s C o r p . , N o r w a l k , CT). DNA a m p l i f i c a t i o n was c a r r i e d out by t h e p r o g r a m of #0 c y c l e s of 94~, l m i n ; 55~, 2min; 72~, 3rain (8). The p r o d u c t was d i g e s t e d w i t h Sac I and Eco RI, and c l o n e d into the p U C 1 8 v e c t o r . To o b t a i n t h e 5 ' - e n d r e g i o n of C P 9 # c D N A ( 5 ' - R A C E ) , lens R N A was r e v e r s e t r a n s c r i b e d as d e s c r i b e d a b o v e e x c e p t for t h e s u b s t i t u t i o n of 100 pmol of pd(N)~ r a n d o m p r i m e r for Not I-d(T)L 8 p r i m e r . The p r o d u c t was c o n c e n t r a t e d and added to D N A t a i l i n g b u f f e r ( B o h e h r i n g e r M a n n h e i m G m b H , G e r m a n y ) . A f t e r i n c u b a t i o n , the product was a d d e d to t h e P C R c o c k t a i l c o n t a i n i n g 100 pmol of e a c h of Not 1-d(T)l ~ p r i m e r and C P P R 2 , and a m p l i f i e d by t h e p r o g r a m as d e s c r i v e d a b o v e . The P C R p r o d u c t s w e r e used as t h e t e m p l a t e s for a s e c o n d P C R r e a c t i o n c o n t a i n e d CPPR1 i n s t e a d of C P P R 2 . The s e c o n d P C R p r o d u c t s of 200191

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5 0 0 b p w e r e d i g e s t e d w i t h E c o RV a n d E c o RI, a n d c l o n e d i n t o p u c l g . R N A w a s e x t r a c t e d f r o m l e n s e s o f SD r a t s ( o n e d a y - o l d , 200 l e n s e s ) , m i c e ( 1 2 9 / S v 3 a n d m i c r o p h t h a l m i c 1 2 9 / S v 3 - E l o ; o n e - w e e k - o l d , t~0 l e n s e s f r o m e a c h ) , a n d n e o n a t a l c h i c k s ( f i v e l e n s e s , f r o m l o c a l p o u l t r y f a r m ) , a n d f r o m v a r i o u s t i s s u e s of p o s t n a t a l SD r a t s ( e a c h t w o g r a m s ) as f o l l o w s : t i s s u e in # M g u a n i d i u m isothiocyanate was disrupted and centrifuged over CsTFA solution. The RNA p r e c i p i t a t e was d i s s o l v e d in, a n d a n a l i q u o t of it w a s f r a c t i o n a t e d o n a 1% a g a r o s e g e l c o n t a i n i n g 2.2 M f o r m a l d e h y d e , a n d t r a n s f e r r e d t o I m m o b i l o n ® - N m e m b r a n e (0.#5~Jm, M i l l i p o r e C o r p . , B e d f o r d , MA). B l o t s w e r e h y b r i d i z e d t o t h e r a n d o m p r i m e d [ 3 2 P ] - l a b e l e d p r o b e s (1× 108 cpm/~Jg) f o r l gh. T h e s e w e r e f i n a l l y w a s h e d w i t h t h r e e c h a n g e s of 0.2× SSC c o n t a i n i n g 0.1% SDS a t 504 (9, 10), a n d e x p o s e d t o X - r a y f i l m w i t h a n s c r e e n f o r 1-#h a t - 7 0 ~ . A c h i c k B - a c t i n g e n e f r a g m e n t w a s p u r c h a s e d f r o m O n c o r Inc., G a i t h e r s b u r g , MD. T h e s e q u e n c e d a t a was handled and analyzed with GENETYX programs (Software Develop. Corp., 3apan). Homology searches were done on all entries of the nucleotide sequence d a t a b a s e s o f E M B L - G D B ( r e l . 2 g . 0 ) a n d G e n B a n k ( r e l . 6 9 . 0 ) , a n d of p r o t e i n s e q u e n c e d a t a b a n k s of S W I S S - P R O T (rel. 19.0) a n d N B R F - P I R ( r e l . 2 9 . 0 ) .

RESULTS AND DISCUSSION Our antiserum

w a s r a i s e d a g a i n s t m o u s e CP9~ t (5), b u t a c D N A l i b r a r y u s e d

w a s m a d e of r a t l e n s b e c a u s e t h e a n t i b o d y r e a c t e d

w i t h a r a t l e n s p r o t e i n of

s l i g h t l y l a r g e r m o l e c u l a r w e i g h t ( 9 5 - 9 6 k D a ) w i t h t h e s a m e s e n s i t i v i t y on W e s t e r n b l o t t i n g . By s c r e e n i n g o f t h e l i b r a r y (~1×106), f o r t y p h a g e c l o n e s e x p r e s s i n g f u s i o n p r o t e i n s w e r e o b t a i n e d . All o f t h e s e c o n t a i n e d t h e s a m e D N A fragment named

o f v a r i o u s l e n g t h s , a n d t h e o n e w i t h t h e l o n g e s t i n s e r t (1.84kbps),

~p9t~.#0, w a s s e l e c t e d a n d m a i n l y u s e d f o r t h e s u b s e q u e n t a n a l y s i s . T h e

fusion protein with ~-galactosidase characterized

a n d r a t CP9t~ w a s p r e l i m i n a r i l y

by W e s t e r n b l o t a n a l y s i s f o l l o w i n g S D S - P A G E (Fig.

1). It w a s

o n l y o b s e r v e d in t h e l a n e l o a d e d w i t h t h e c e l l i n f e c t e d w i t h ~p9t~.t¢0 i n d u c e d w i t h IPTG a n d h a r v e s t e d used for detection, estimated

just b e f o r e s p o n t a n e o u s lysis. With a n t i - C P 9 4

the apparent

as 190-200kDa,

antibody

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

indicating that the cDNA insert of the clone seemed

t o e n c o d e a p o l y p e p t i d e w i t h 8 0 - 9 0 k D a ( a s s u m i n g t h e / t - g a l a c t o s i d a s e p a r t of the fusion protein to be l]0kDa) which corresponds to 84-94% of mature

rat

CP9t~ s e q u e n c e .

coding

capacity

However the insert size was

of it w a s c a l c u l a t e d

1.84kbps, a n d t h e m a x i m u m

at 68kDa polypeptide.

We c a n n o t c o n c l u s i v e l y

explain this discrepancy at present, but this result suggests that the c o n f i g u r a t i o n of t h e C P 9 4 m o l e c u l e is f a r d i f f e r e n t f r o m a g l o b u l a r s h a p e . T h e anti-chick

CP97 antibody also reacted

w i t h 2p9t~.40 l y s a t e , but b a n d s w i t h

l o w e r m o l e c u l a r w e i g h t s w e r e o b s e r v e d , p r o b a b l y r e p r e s e n t i n g d e g r a d e d f o r m s of the CP9# fusion protein. N o r t h e r n b l o t a n a l y s i s i n d i c a t e d t h a t t h e c l o n e d i n s e r t ~p94.40 h y b r i d i z e d t o a m R N A of 2 . 1 - 2 . 3 k b l e n g t h in t h e l a n e l o a d e d w i t h R N A f r o m r a t l e n s (Fig. 2). No s i g n a l w a s d e t e c t e d

in t h e l a n e s of 1RNAs f r o m b r a i n , skin,

heart, kidney, lung, and liver, thus confirming that the expression of the

192

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o-, m cJ

P'~ o-, ca C~

:

.h