1993 Au Molecular Cloning and Sequence Analysis of the CDNA for Ancrod C. Rhodostoma

8/17/2019 1993 Au Molecular Cloning and Sequence Analysis of the CDNA for Ancrod C. Rhodostoma

1/4

Biochem.

J. (1993) 294

387 390 (Printed

in Great

Britain)

M o l e c u l a r

c l o n i n g

a n d

s e q u e n c e

a n a l y s i s

o f t h e

c D N A

f o r

a n c r o d ,

a

t h r o m b i n - l i k e

enzyme f r o m

t h e

venom o f C a l l o s e l a s m a

r h o d o s t o m a

L o - C h u n A U , * t §

S h w u - B i n L I N , t

J e n g - S h e n g

C H O U , 4

G o - W a i

T E H , * K u o - J u i

C H A N G t

a n d

C h i a - M i n

S H I H t

* D e p a r t m e n t o f M e d i c a l

R e s e a r c h , V e t e r a n s G e n e r a l

H o s p i t a l - T a i p e i , T a i p e i , t S c h o o l

o f M e d i c a l

T e c h n o l o g y ,

N a t i o n a l

Y a n g M i n g

M e d i c a l

C o l l e g e ,

T a i p e i ,

a n d

t G r a d u a t e

I n s t i t u t e

o f M e d i c a l

T e c h n o l o g y , D e p a r t m e n t

o f

C l i n i c a l

P a t h o l o g y , C o l l e g e

o f

M e d i c i n e ,

N a t i o n a l T a i w a n

U n i v e r s i t y ,

T a i p e i , T a i w a n ,

R e p u b l i c

o f

C h i n a

T h e

1 . 5 4 k b

cDNA

f o r

a n c r o d ,

a


enzyme,

wa s

c l o n e d f r o m a AZAP

cDNA

l i b r a r y d e r i v e d f r o m t h e venom

g l a n d s o f C a l l o s e l a s m a

( A g k i s t r o d o n ) r h o d o s t o m a . T h e

cDNA

sequence r e v e a l s t h a t

a n c r o d

i s

s y n t h e s i z e d

as

a

pre-zymogen o f

2 5 8 a m i n o a c i d s , i n c l u d i n g

a p u t a t i v e

s e c r e t o r y

p e p t i d e o f

1 8

a m i n o a c i d s a n d

a

p r o p o s e d zymogen p e p t i d e o f 6 a m i n o - a c i d

r e s i d u e s . T h e a m i n o - a c i d

sequence

o f t h e

p r e d i c t e d

a c t i v e

f o r m

o f

t h e e n z y m e

e x h i b i t s

a

h i g h

d e g r e e

o f

sequence s i m i l a r i t y

t o

t h o s e o f m a m m a l i a n

s e r i n e

p r o t e a s e s ( t r y p s i n a n d

p a n c r e a t i c

k a l l i k r e i n ) a n d o t h e r


e n z y m e s ( b a t r o x o b i n

a n d

I N T R O D U C T I O N

S n a k e venoms

a f f e c t

b l o o d

c o a g u l a t i o n

a n d

p l a t e l e t

f u n c t i o n s i n

a

c o m p l e x

manner

[ 1 ] .

T h e

venom o f

t h e

s n a k e C a l l o s e l a s m a

r h o d o s t o m a

( f o r m e r l y k n o w n as

A g k i s t r o d o n

r h o d o s t o m a )

c o n t a i n s

b o t h

p l a t e l e t - a c t i v a t i n g

a n d

p l a t e l e t - i n h i b i t o r y

com-

p o n e n t s ,

a

h a e m o r r h a g i c p r o t e i n a n d

a


e n z y m e

[ 1 - 7 ] . T h r o m b i n - l i k e

e n z y m e s

[ 8 - 1 0 ]

are

s e r i n e

p r o t e a s e s , w h i c h

b e l o n g

t o

t h e

t r y p s i n

f a m i l y

[ 1 1 ] .

I n

c o n t r a s t w i t h

t h r o m b i n ,

w h i c h

c o n v e r t s

f i b r i n o g e n

t o f i b r i n

b y r e m o v i n g

f i b r i n o p e p t i d e s

A

a n d B , a t h r o m b i n - l i k e

e n z y m e r e l e a s e s

o n l y f i b r i n o p e p t i d e A

f r o m

f i b r i n o g e n

[ 1 2 , 1 3 ] .

P a r e n t e r a l a d m i n i s t r a t i o n

o f

t h e

enzyme

causes t h e

c o n v e r s i o n

o f

f i b r i n

i n t o a

f i b r i n d e r i v a t i v e t h a t

i s

r a p i d l y d e g r a d e d a n d

e l i m i n a t e d

t h r o u g h a f i b r i n o l y t i c

process i n

t h e b l o o d s t r e a m

[ 1 2 ] . A n c r o d

i s

a h i g h l y a c t i v e


e n z y m e p u r i f i e d f r o m t h e venom o f

C a l l o s e l a s m a

r h o d o s t o m a

[ 1 3 ] .

S i n c e

a n c r o d

h a s

b e e n

c o m m e r c i a l l y

a v a i l a b l e

f o r many

y e a r s , numer ous

e x p l o r a t o r y s t u d i e s on i t s

c l i n i c a l

a p p l i c a t i o n

h a v e

b e e n

r e p o r t e d [ 1 4 ] . T h e

r e s u l t s

h a v e

i n d i c a t e d

c o n s i d e r a b l e

p o t e n t i a l

v a l u e f o r

t h i s

p r o t e i n

i n

t h e t r e a t m e n t o f

v a r i o u s

t h r o m b o - e m b o l i c

s t a t e s , a n d

i n

t h e

p r e v e n t i o n

o f

t h r o m b o s i s .

H o w e v e r , t h e m o l e c u l a r

c h a r a c t e r i s t i c s o f a n c r o d

h a v e n o t b e e n

d e s c r i b e d .

P r e v i o u s l y ,

we

h a v e c l o n e d a n d

a n a l y s e d

t h e

cDNA

f o r a

p l a t e l e t - a g g r e g a t i o n

i n h i b i t o r

a n d f o r a

h a e m o r r h a g i c

p r o t e i n

t h a t

were

d e r i v e d

f r o m

t h e

v e n o m - g l a n d

cDNA

l i b r a r y

o f C a l l o s e l a s m a

r h o d o s t o m a

[ 1 5 ] . H e r e ,

we

r e p o r t

t h e

c l o n i n g

a n d

n u c l e o t i d e - s e q u e n c e

a n a l y s i s

o f

t h e

cDNA

f o r

a n c r o d ,

a n d

t h e

d e d u c e d

a m i n o - a c i d

sequence.

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

M E T H O D S

I s o l a t i o n o f c D N A

c l o n e s

f o r a n c r o d

A

s e t

o f

cDNAs

d e r i v e d f r o m

a

p o l y ( A ) +

RNA

p r e p a r a t i o n

o f

t h e

venom

g l a n d s

o f

C a l l o s e l a s m a

r h o d o s t o m a were

c l o n e d

i n t o

t h e

Uni-ZAPTMXR

v e c t o r

[ 1 6 ]

b y i n s e r t i n g

t h e cDNAs i n t o

f l a v o x o b i n ) .

K e y

a m i n o - a c i d

r e s i d u e s ( H i s 4 3 , A s p 8 8 ,

S e r l 8 2 a n d

A s p 1 7 6 )

t h a t are t h o u g h t t o b e i n v o l v e d

i n

t h e s u b s t r a t e c l e a v a g e

a n d i n

t h e s u b s t r a t e - b i n d i n g

r e a c t i o n a r e c o n s e r v e d .

A n c r o d

c o n t a i n s

1 3 c y s t e i n e r e s i d u e s .

B a s e d

o n a l i g n m e n t w i t h

t h e

a m i n o - a c i d

sequences o f

t r y p s i n

a n d

b a t r o x o b i n , s i x

d i s u l p h i d e

b r i d g e s ca n b e

p r e d i c t e d t o b e

p r e s e n t

i n

t h e a n c r o d

p r o t e i n . T h e

e x i s t e n c e o f a

f r e e

c y s t e i n e , w h i c h

c h a n g e s t h e

common

sequence

s u r r o u n d i n g

t h e

S e r 1 8 2 a c t i v e s i t e

f r o m

G l y - A s p - S e r - G l y - G l y - P r o

t o

C y s - A s p - S e r - G l y - G l y - P r o ,

i s

u n u s u a l

f o r

a s e r i n e

p r o t e a s e .

t h e E c o R I a n d

X h o I s i t e s

o f

t h e

Uni-ZAPTMXR

p h a g e

DNA.

F o r

c o n v e n i e n c e ,

t h e

Uni-ZAPTXR

v e c t o r was e x c i s e d i n

v i v o

t o

g e n e r a t e a p B l u e s c r i p t S K - p h a g e m i d cDNA

l i b r a r y b y co-

t r a n s f e c t i n g

t h e Uni-ZAPMXR v e c t o r a n d

an

R 4 0 8

h e l p e r

p h a g e

[ 1 7 ]

i n t o E s c h e r i c h i a c o l i

X L 1 - B l u e h o s t

c e l l s a c c o r d i n g t o

t h e

m a n u f a c t u r e r s

i n s t r u c t i o n s

( S t r a t a g e n e ,

L a

J o l l a ,

C A , U . S . A . ) .

E . c o l i

X L 1 - B l u e was t h e n

i n f e c t e d

w i t h

t h e

r e s c u e d

p h a g e m i d .

T h e i n f e c t e d

b a c t e r i a

were p l a t e d o u t

on

t o L u r i a

b r o t h / a m p i c i l l i n p l a t e s .

A m p i c i l l i n - r e s i s t a n t

c o l o n i e s

were

t r a n s -

f e r r e d on

t o

n i t r o c e l l u l o s e membrane a n d

p r e p a r e d

f o r

c o l o n y

h y b r i d i z a t i o n

[ 1 8 ] .

A

2 0 m e r i c

o l i g o n u c l e o t i d e

p r o b e

( s e e F i g u r e

1 )

was


a c c o r d i n g

t o

t h e

n u c l e o t i d e

sequence

o f

a

b a t r o x o b i n cDNA

[ 8 ] . T h e

p r o b e

wa s

3 2 P - e n d - l a b e l l e d w i t h T 4

p o l y n u c l e o t i d e k i n a s e

a n d

[ y - 3 2 P ] A T P

[ 1 9 ] .

T h e

m e m b r a n e

r e p l i c a s were

p r e - h y b r i d i z e d

a t 6 5

° C f o r

3

h

i n


b u f f e r

[ 0 . 4 5

M

s o d i u m

c h l o r i d e , 0 . 0 4 5

M

s o d i u m

c i t r a t e , 0 . 0 2 M

p o t a s s i u m

p h o s p h a t e , pH 6 . 8 , 0 . 1

( w / v )

S D S ,

0 . 0 2 0

( w / v )

f i c o l l a n d

0 . 0 2 ( w / v )

p o l y v i n y l p y r r o l i d o n e ]

p l u s

100

g / m l

s h e a r e d

a n d

d e n a t u r e d

h e r r i n g - s p e r m

DNA.

T h e

m e m b r a n e

r e p l i c a s

were

t h e n

h y b r i d i z e d a t

5 4 °C

f o r 8 h i n

3 0 m l o f


b u f f e r

c o n t a i n i n g

5

p m o l

o f t h e l a b e l l e d

o l i g o -

n u c l e o t i d e

p r o b e .

S u b se qu e nt w a s h es o f t h e

h y b r i d i z e d mem-

b r a n e f i l t e r s were c a r r i e d o u t i n

6

x

SSC

1

x

SSC

i s

0 . 1 5

M

N a C l / 0 . 0 1 5

M s o d i u m

c i t r a t e )

a n d

0 . 1

( w / v ) SDS as

f o l l o w s :

f o u r

t i m e s a t 4

° C

f o r

5 m i n

e a c h ,

once a t room

t e m p e r a t u r e

( 5 m i n ) a n d once a t

5 0 ° C

( 2

m i n ) .

T h e

p o s i t i v e

c l o n e s were

d e t e c t e d

b y

a u t o r a d i o g r a p h y ,

a n d

were f u r t h e r

p u r i f i e d

b y

i s o l a t i n g

s i n g l e

c e l l

c o l o n i e s .

D e t e r m i n a t i o n

o f

t h e n u c l e o t i d e

s e q u e n c e

o f t h e

c D N A i n s e r t

T h e

p h a g e m i d s

o f

p o s i t i v e

c l o n e s

were

p u r i f i e d b y

a l k a l i n e

l y s i s

o f

t h e

h o s t

c e l l ,

f o l l o w e d

b y

e q u i l i b r i u m

c e n t r i f u g a t i o n

i n

a

C s C I / e t h i d i u m - b r o m i d e

g r a d i e n t

[ 1 8 ] .

The n u c l e o t i d e

sequence

o f

t h e

cDNA

i n s e r t was d e t e r m i n e d

b y

t h e

d i d e o x y

method

u s i n g

t h e

w a l k i n g p r i m e r

s t r a t e g y ( F i g u r e

2 ) .

§

T o

whom

c o r r e s p o n d e n c e

s h o u l d b e a d d r e s s e d .

T h e

n u c l e o t i d e

s e q u e n c e

r e p o r t e d

i n

F i g u r e

3

w i l l

a p p e a r

i n t h e GenBank

D a t a b a s e u n d e r

a c c e s s i o n

n o .

L 0 7 3 0 8 .

3 8 7

i o c h e m . J .

1 9 9 3 )

2 9 4 ,

3 8 7 - 3 9 0

P r i n t e d

i n

G r e a t

B r i t a i n


2/4

L . - C .

A u a n d o t h e r s

O l i g o n u c l e o t i d e

p r o b e

G AA

CTO

I

G T C

AT T OGA

G G T

G AT

G A k

T O G C

Glu

Lo u

V a l I l e Gly G ly Asp O lu

C ys

Asp

- 1 1

7

F i g u r e

1

O l i g o n u c l e o t i d e

p r o b e ( 2 0 m e r ;

b o x e d )


a c c o r d i n g

t o

t h e n u c l e o t i d e

s e q u e n c e

o f

t h e

N - t e r m i n a l

a m i n o - a c i d

r e s i d u e s

o f

b a t r o x o b i n

[ 8 ]

N h e l

B a l l

P v u l l

B s p M I I

4 0 0

N c o l

P s t I

H p a l

EcoRI

BamHI

P v u l l D r a l

Smal

BamHI

I

8 0 0

2

F i g u r e

2

P a r t i a l

r e s t r i c t o n

m a p

a n d

s e q u e n c i n g

s t r a t e g y

u s e d t o

o b t a i n

t h e

n u c l e o f t d e

s e q u e n c e

o f

t h e

cDNA

c l o n e

J - 5

A r r o w s i n d i c a t e

d i r e c t i o n s a n d

s e q u e n c e d r e g i o n s .

T h e

l o n g e s t

o p e n

r e a d i n g

f r a m e

i s

s h o w n

a s a

f i l l e d

b o x .

RESULTS AND

D I S C U S S I O N

S c r e e n i n g

o f a

cDNA

l i b r a r y

Based o n t h e n u c l e o t i d e

s e q u e n c e

o f

t h e N - t e r m i n a l

a m i n o - a c i d

r e s i d u e s

of

batroxobin

[ 8 ] ,

a

2 0 m e r i c

o l i g o n u c l e o t i d e

was

s y n t h e s i z e d ( s e e F i g u r e 1 )

and wa s u s e d

a s

a

h y b r i d i z a t i o n p r o b e

i n

t h e

s c r e e n i n g

o f

a

p h a g e m i d - b a s e d

cDNA

l i b r a r y

as

d e s c r i b e d

i n

t h e M a t e r i a l s and

metho ds

s e c t i o n . T h r e e

p o s i t i v e

c l o n e s

( d e s i g n a t e d

J - 2 ,

J - 3

and

J - 5 )

were

o b t a i n e d

from

t h e

s c r e e n i n g

o f

about 1000

c o l o n i e s .

T h e s i z e of

t h e

cDNA

i n s e r t

i n

e a c h

p o s i t i v e

c l o n e was

determined

b y

PCR

u s i n g

a

p a i r

o f

p r i m e r s f l a n k i n g

t h e

c l o n i n g

s i t e s

o f

t h e

p B l u e s c r i p t S K - p h a g e m i d

DNA.

The

r e s u l t s

i n d i c a t e d

t h a t

J - 2 ,

J - 3 and

J - 5 were

i n d e p e n d e n t

and

o v e r l a p p i n g

c l o n e s

c a r r y i n g

cDNA

i n s e r t s

o f

1 . 4 ,

1 . 3

and

1 . 5

k b

r e s p e c t i v e l y .

N u c l e o t i d e - s e q u e n c e a n a l y s i s

Clone

J - 5 ,

which

c a r r i e s t h e

l o n g e s t

cDNA

i n s e r t ,

was

c o m p l e t e l y

s e q u e n c e d ( F i g u r e 2 ) .

Most

r e g i o n s

o f

t h e cDNA

were

s e q u e n c e d

i n

both d i r e c t i o n s . On

a n a l y s i s

o f t h e

n u c l e o t i d e

s e q u e n c e

o b t a i n e d ,

a

l o ng o p e n r ea d i n g

frame

wa s

o b s e r v e d

( F i g u r e

2 ) .

I n

t h i s

o p e n r ea d in g f r a m e ,

t h e

t r a n s l a t i o n - i n i t i a t i o n s i t e

i s

a s s i g n e d

t o

t h e f i r s t

methionine

codon

a t

n u c l e o t i d e s

1 7 8 - 1 8 0 ,

which

i s

f o l l o w e d

b y

an

u n i n t e r r u p t e d

c o d i n g

r e g i o n ,

which

p u t a t i v e l y

codes

f o r 258

amino-acid r e s i d u e s

( F i g u r e

3 ) .

The 3

n o n - c o d i n g

r e g i o n

c o n t a i n e d a

p o l y ( A ) + s e q u e n c e

and

a

p u t a t i v e

p o l y a d e n y l a t i o n s i g n a l s e q u e n c e

AATAAA

( n u c l e o t i d e s

1 5 1 9 -

1 5 2 4 ) ,

which

was l o c a t e d a t

a

s i t e

1 4

n u c l e o t i d e s

u p s t r e a m

from

t h e

p o l y ( A ) + s e q u e n c e ( F i g u r e 3 ) .

Two

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

s i t e s ,

A s n - X a a - T h r ,

were l o c a t e d a t

amino-acid

r e s i d u e s 1 4 8 - 1 5 0

and 229-231.

Ancrod

i s

known t o

b e a

g l y c o p r o t e i n

[ 2 0 ] .

A m i n o - a c i d

s e q u e n c e

a n a l y s i s

T h e

amino-acid

s e q u e n c e

deduced from

t h e

c o d i n g

r e g i o n

o f

t h e

1

3 1

T G A

C TC T OC C AG

C GT T TG AGA

TT G

TT G

GC C

AC C

C AG C OG

C T T

AAT

T TG

ATC

AAA

TAA

AG T

6 1

9 1

GC T

GC T T WA

TCA

AG A AG T

CT C CTC

T? O

AOTT T

CTG

AT T

AGA

TT G

T Coo

T T

CTC

MG

1 2 1

1 5 1

G 0 T

AAG TT S G 0 G AC T

G 0 0

AT C TT A

C AG O C A

M

AMC

TrCTOr

TO C M G A 0T

GA A

G C T AT G

1 8 1 2 1 1

4

G T G C T G

AT C

AG A GT G

C T A

GC A

AAC

C TT G TG

ATA C T A

CM

CT ? TCT

T AC

GC A

CAA AA G

TC T

va l

l e u

ile

arg

va l

l e u

ala

a n l eu v a l

il e l eu gl n le u

m er

tyr

al a

gl n

ly e

se r

2 4 1 2 7 1

- 4

T C T G AA

CTG IGTC

Arr

GC A

OG T G AT G AA TG T AAC AT A AA T GA A

CAT

C G T

T T C

C TT G TA GC C

ser

gl u

le u

va l

il e

gl y

g l y

asp

gl u

cys

as n

i l e

aen gl u h is

ar g

ph e

le u

val al a

3 0 1 - 1 1 3 3 1 1 7

TT G TA T G AC

AG T

AC G

AC T

C GG AAT

m T T C TC

TG T G GT G GG

G TT ITO AT C C AT

C C G GA A

TG G

lo u ty r

as p

se r

t hr t hr

arg

as n ph e

l e u

cys

gl y gl y

va l

l e u il e h is

pro gl u

tr p

3 6 1 3 9 1 3 7

G T G

AT C AC T

O CT

AAA

C XC TG C

AAC

AAG

AM AGT AT G

G TC C TA

TA C

C TT

G O T AAG C AT

AAA

v al

i l e

th r al a ly s his

cy s

aen

l ye l ye

me r

m e t

va l le u t y r

l e u gl y

l y s his

1 y m

4 2 1 4 5 1

9 7

C AA AG T G TA AM

T r r

G AC

GA T G AG C AG

G M A AG A T TC

C C A AAG

G AG A AG C AC

TT T

AT T

C GC

gl n m er

va l

ly s

ph e a sp a sp gl u

gl n

gl u ar g ph e

pro

ly e gl u ly e hi s ph e ile

ar g

4 8 1

*77

T G T AAC AAA CC C C GT ACC

AG A

TG G

GG C

G AG G AC

AT C AT G

T T G

ATC

AG

CT

AAC AAA

C CT

cy m as n

l y e

pro ar g

th r ar g

t rp gl y gl u as p ile

m e t l e u

i l e

ar g le u asn ly s pro

5 4 1 5 7 1 9 7

G T T AAC AAC AG T

G AA

C AC AT C

GC T

C CT C T C AMC T T I C C T TC C AAC C C T C C C AT T GT G GG C

va l as n ao n

m e r

gl u h is i l e al a pro le u ser

le u

pro se r as n p ro p ro i l e va l

g l y

6 0 1

6 3 1 1 1 7

T C A

G TT

TG C

CG T G TT ATG

GG A TG G GO C TC A AT C AAT

AAA

T AT AT A

GA C

G T T

TT O C C C

GAT

me r

v al cy s ar g v al m et gl y

t rp g ly

m e r il e ag n l y s

ty r

i l e

asp

va l l e u pro

am

6 6 1

6 9 1

1 3 7

G AA C C T

C GT T GT

GC T AAT

A?

AAC

C TG

T AC

AAT TAC

AC G

G TO

TG T

C O T

GG A GT T

TT

C C A

gl u

pro

arg

cy s

al a as n

ile

0sn

le u

ty r

asn

ty r th r

va l

cy s

arg

gl y

vo l

ph

pro

7 2 1

7 5 1 1 5 7

AG G AT A C C A AA G A MA

AG C

AAA AT A TTG TG T G CA

GG T G AC C TG

C AA O GA CGC C TA GAT

TC A

ar g i l e

pro ly s l y s

se r

ly e

i l e l e u

cy s al a gl y a sp le u

gl n

gl y ar g

l e u

asp

m e r

7 8 1

8 1 1

1 7 7

TO T

C AC

TG T

G AC T CT

G G G

GO A

C C T

C TC AT T

T GT AG T

G AA G AA TT C

C AT

GOC AT T

G TA T AT

cy s h is cy s as p

se r

gl y gl y pro le u i l e

cy s smr

gl u gl u ph e hi s gl y ile va l

ty r

8 4 1

8 7 1 1 9 7

C G G

G GA C CC AAT C C T

T G T 0C C

C AA

C C A GA T AAG C C T

GC C C T C TAC AC C AAC ATC TTC GA T

ar g

gl y

pro a n pro

cy s al a gl n pr o

a sp l ys

pro al a

l a u

ty r

th r

as n i l e

ph e

asp

9 0 1

9 3 1 2 1 7

C AT C TT

C AC

TO G

AT C

C TT

AG C

AT T

AT G G C A

GG A

AAT GC A AC T TG C T AT C C A T AA

AAC C T T

hi s le u

hi s t rp

i l e l eu me r

il e m et al a glylaon

o l a

thrcys

ty r

pr o

9 6 1

9 9 1 Z 3 4

TT G

A AA T AG TT A

AG T

O G A GM

AAT G TA AC A

T A T

T AG T AA AT C

TC T

T C T

ATA

TC C ST G C AT

1 0 2 1

1 0 5 1

T G G AAC

AT A

TT C

C C A GC T

GT A AG C

C? ? T?? AG A

C TC

AM TAG

G AC

TA C C ? T T O G G

MT

AAG

1 0 8 1 1 1 1 1

M A G TG C

T C A AM

TA G

TG C TO C A GG G AT

C AT

??C

C C A

P T T

AAT

TT C

AG T ?? A

AM

C AG

TO

1 1 4 1

1 1 7 1

CC

TAG

AT T G G A

G O A

C TG T AG

G O T

TAG

G T

CAA AAT T?? CT G

AC T

C T A

AM TO G A C C

1 2 0 1

1 2 3 1

A ? ?

C C A

AT C ?? T TAA

C C T C TG

AAT

A

TTC

AAT TTC ? T C AG C? C TG

G 0 C AG AT C T O C

1 2 6 1

1 2 9 1

TC T

G A O C ? ?

GT C ST C

T G O

C AG

AC A T TT C AT

TA C C C A

OC T A G O TA A

C AT

C AT CM TO C

TAG

1 3 2 1

1 3 5 1

AAT A?T ? ? T ??C TA T TG O TA T ?? C T O T G G C

A? ?

TA C AAT AC G C TC

? T A

T O MT T AT C CA

1 3 8 1 1 4 1 1

G TC AC C

C C A

CA M

AC A T AT

AC A

T AT

C C C

CO G GT C C C A C T T T G

C C T A M A AM G A T C C C

A G A

1 4 4 1

1 4 7 1

? ? A

AC C

CCC

CAT

TT C

CCA

AT C AC T

A AA T AG

AAT

C T T I T I

AG A

AT O

AT G

TT T TC A WT T AAA

1 5 0 1 1 5 3 1

TT C

T TA

G G T

AT C

C AC AAC

AAT AAA

T TG

T AT

AAA T CA T CA

AM AM

AM

AM AM

M A M A

F i g u r e

3

N u c l e o t i d e

s e q u e n c e

o f

t h e a n c r o d c O N A

J - 5

a n d 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 a n c r o d

p r e c u r s o r

p r o t e i n

T h e

n u c l e o t i d e

r e s i d u e s

a r e

n u m b e r e d

o n

t h e

l e f t

a n d

t h e

a m i n o - a c i d

r e s i d u e s

a r e

n u m b e r e d

o n

t h e

r i g h t .

T h e

a m i n o - a c i d

n u m b e r i n g

s t a r t s a t t h e

N - t e r m i n a l

a m i n o a c i d , v a l i n e o f a n c r o d .

T h e

p u t a t i v e p r e - p e p t i d e a n d

p r o - p e p t i d e c o n s i s t o f a m i n o a c i d r e s i d u e s

- 2 4

t o -7 a n d -6

t o

-1

r e s p e c t i v e l y .

A r r o w s

i n d i c a t e

p r e d i c t e d

c l e a v a g e

s i t e s . A s t e r i s k s i n d i c a t e

p u t a t i v e a m i n o -

a c i d

r e s i d u e s t h a t

a r e i n v o l v e d

i n

c a t a l y t i c

a c t i o n . T h e

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

s i t e s

ar e s h o w n

i n

b o x e s

a n d

t h e

p o l y a d e n y l a t i o n - s i g n a l s e q u e n c e ,

A A T A A A , i s

u n d e r l i n e d .

J - 5

cDNA

( F i g u r e 3 )

was

c o m p a r e d w i t h t h a t o f t h e b a t r o x o b i n

precursor ( s e e

F i g u r e 4 )

[ 8 ] . B a t r o x o b i n i s

a t h r o m b i n - l i k e

e n z y m e

t h a t i s

d e r i v e d f r o m

s n a k e

venom o f B o t h r o p s atrox

m o o j e n i .

O n

a l i g n m e n t

u s i n g t h e m e t h o d

o f

P e a r s o n a n d Lipman

[ 2 1 ] ,

60

sequence

i d e n t i t y

( 7 0 0

s i m i l a r i t y

c o n s i d e r i n g

con-

s e r v a t i v e

s u b s t i t u t i o n s )

was

found b e t w e e n

t h e

J - 5 - d e r i v e d

a m i n o -

a c i d

s e q u e n c e

and

t h a t o f

b a t r o x o b i n

[ 8 ] .

T h i s

i m p l i e s

t h a t t h e

3 8 8

..

I

I

a

m

m


3/4

M o l e c u l a r

c l o n i n g

a n d

s e q u e n c e a n a l y s i s

o f c D N A f o r a n c r o d

t i d e

- p e p t i d e

- 2 4 - 7

1

XV

L

I R

V L A

N

L

V

I

L

Q

L S Y A

Q

8

L

X

V

L I R

V L A N L L

I

L

Q V 8

Y

A

Q

K

8

8

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L

2 0

V

I

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D 8

C N I

N

b

N

R

P L V A

L

Y D

ST

T

R

N

r

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a

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A Y y

8 P R

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C

GX

V I

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N I N

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8

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K

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A

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I

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8

Y

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r

s

QIKIII Q

N

8 0

1 0 0

K

R T R

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D I

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L

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N

N

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N I

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P L

S

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N

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D K D

I

L

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L

D R

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P

L L

P

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I

N

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D

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V

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8

S

H I A

P L

S P

N W K

e

L D R D

I

A

L

L

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P

I

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Y I

H P V

C L P

N

S N T

L

N

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D I

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A

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A

I

8

L

P

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8

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a

N

H

D

I

A L

I

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Q

A P

L N

Y

T

F Q

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I

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12 0

S

N

P P I V

G

S

V

C

R

VK

W

G

S

N K Y I D V

S N

P

P

S VS

C

R I G

W

A

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S

D

T

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V

S

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S

I T P V T

D R

T A AK

L

H

G

F K

G

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T

a

W

a

N

R

R

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S

V

T

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A

S

A

G

T

Q

C

L

I

S

G

W

N

T K

S S G

T

S

S

K

D

T

S T

I

Y

T N

C

N

T

GNG

S

K

O

B

I

1 40

1 60

L P D P

R

C

A

N

I

N L Y

N Y

T V

C R

G

Z

P R I

P

K

K

Y

P

D V

P

H

C A N I

N

L

P N N T

V

C R

Y N

G

P

F

P D

V

P H C A N I N

L

L D D V

C

K

P

a

Y P

L

L P 8

A

3

Q

P

S

V LQV V N

L

P L V

R

P

C

K

A

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I R

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P D V

L

K

C L

K

P I L

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S

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S A Y

P

a o

I

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Q

I

L

Q

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8

C

Q

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D

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1 8 0 .

S K I L

C A

G

L

Q

G

L D

S C H C

D

S a

G

P

L

I

C

A K T L C

a

v

L

Q

a G

I

T

C

G

D

SGG

P

L

I

C

Y R T L

C

A

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L

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G

c

I D

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S

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L

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A

G

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P G G

K

G

D

C

C

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7

F

V

K

S

N F C A

G

Y L

G G K D

S C

Q

G

S G G P

V

V C

Q

V

C

A

G

K

G G K

D

A

C K G D

S

G

Q

P

L

V C

2 0 0

S E1 Z H

G

V

Y

RG

N P C A

Q P D

K

A L

Y

T N I

N

G

Q

FQ

G I L S

I

G S

D P C A R

P

R

K

A F Y

T

K

V

N G

Q

FQ

G I

V

Y

I G

8 H P C G

Q

8 R K P

G I Y T

K

P Y N

N

R

W Y

Q

N

I V S W

G G

C

D R N

GK

G

F Y T H V

S

G K L

Q

G I V

S

G S

G

A Q

KK

G

V

Y T

R

V

K H N G

N

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V G I T S G

2

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A

R R

Q

P

G V

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2 2 0

F

D

H

L HNI L

S

I

N

G

N

A

T

Y P

F

Y

L P

Q

S

I A

GKA

C

P

D

Y

N A

W

I

Q

S

I A G N

T

A

T

C L P

F

R L

K K W I K

V

I

D

R

L G

S

C

N

Y

V

S

W I

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8

D

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8

P

A

a n c r o d i s v a l i n e

[ 2 0 ] . T h e h y d r o p h i l i c p e p t i d e ( a m i n o

a c i d s

-6

t o - 1 )

i s p r e s u m a b l y a z y m o g e n p e p t i d e .

F i g u r e 4

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

a n c r o d

a n d o t h e r m a t u r e s e r i n e

p r o t e a s e s [ 9 , 2 2 , 2 3 ] .

B a s e d

o n

s e q u e n c e

s i m i l a r i t y , w e c o u l d d e d u c e

t h e c a t a l y t i c

a m i n o - a c i d r e s i d u e s

t o

b e

H i s 4 3 , A s p 8 8 a n d S e r 8 1 2 ( F i g u r e

4 ) .

T h e l o c a t i o n

o f

A s p 1 7 6 a t

a p o s i t i o n e q u i v a l e n t

t o

t h e b o t t o m o f t h e

s u b s t r a t e - b i n d i n g

p o c k e t i s

c o n s i s t e n t

w i t h

t h e

f a c t

t h a t

a n c r o d

c l e a v e s

a

f i b r i n o p e p t i d e

A f r a g m e n t

f r o m

f i b r i n o g e n

a f t e r

t h e

p o s i t i v e l y

c h a r g e d a r g i n i n e r e s i d u e

[ 1 3 ] .

A n c r o d

c o n t a i n s 1 3

c y s t e i n e

r e s i d u e s .

B a s e d o n

c o m p a r i s o n

o f

t h e a l i g n e d s e q u e n c e s o f

b a t r o x o b i n ,

f l a v o x o b i n

a n d

t r y p s i n ,

s i x

p o s s i b l e

d i s u l p h i d e b r i d g e s

a p p e a r

t o

b e

C y s 7 _ C y s I 4 1 ,

C y s 2 8 - C y s 4 4 ,

C y s 7 8 _ C y s 2 3 2 , C y s 1 2 0 _ C y s 1 8 8 , C y s 1 5 2 _ C y s t 6 7

a n d

C y s 7

- C y s 2 0 3

T h e

e x i s t e n c e

o f a f r e e

c y s t e i n e

( C y s 1 8 0 ) ,

w h i c h

c h a n g e s

t h e

common

s e q u e n c e

s u r r o u n d i n g

t h e

a c t i v e

s i t e

S e r 1 8 2 f r o m

G l y -

A s p - S e r - G l y - G l y - P r o t o

C y s - A s p - S e r - G l y - G l y - P r o ,

i s

u n u s u a l

f o r

a

s e r i n e

p r o t e a s e .

To e n s u r e t h a t a m i n o - a c i d r e s i d u e s

u n i q u e

t o a n c r o d

( s u c h

a s

C y s 1 8 0 , L y s 4 2 , A r g t 7 4 )

f o u n d

i n

t h e

s e q u e n c e

a l i g n m e n t ( F i g u r e 4 ) w e r e n o t a r t e f a c t s

d e r i v e d

f r o m t h e cDNA

c l o n i n g

p r o c e s s ,

a n

i n d e p e n d e n t c l o n e

J - 2

w a s a l s o

s e q u e n c e d .

T h e

s e q u e n c e

o f

J - 2

w a s

c o n s i s t e n t

w i t h

t h a t

o f

t h e

J - 5 c l o n e .

T h e r e p l a c e m e n t o f

G l y 8 0

b y

a

c y s t e i n e r e s i d u e

i n t h e common

s e q u e n c e a r o u n d t h e s e r i n e r e s i d u e

o f

t h e a c t i v e s i t e d o e s n o t

s e e m t o d e c r e a s e t h e t h r o m b i n - l i k e

p r o t e o l y t i c

a c t i v i t y

o f a n c r o d

c o m p a r e d

w i t h t h a t o f b a t r o x o b i n

[ 8 ] .

F i n a l l y ,

w e

s p e c u l a t e

t h a t a n c r o d

may

a l s o

p o s s e s s

a

c y s t e i n e

p r o t e a s e - l i k e

a c t i v i t y .

C y s t e i n e p r o t e a s e s

s h a r e

s o m e

s i m i l a r i t i e s

o f r e a c t i o n m e c h a n i s m

w i t h

s e r i n e

p r o t e a s e s

[ 2 6 ] ,

b u t

t h e y

a r e

g e n e r a l l y

a c t i v e i n

a h i g h e r

pH

r a n g e .

T h e - S H

g r o u p

o f a f r e e

c y s t e i n e

r e s i d u e

p l a y s

a

r o l e

t h a t

i s s i m i l a r

t o

t h a t o f t h e

-OH

g r o u p

o f t h e a c t i v e - s i t e

s e r i n e .

C y s 1 8 0 ,

w h i c h

i s

l o c a t e d

c l o s e t o

t h e

c a t a l y t i c c a v e ,

may

b e

c o u p l e d

w i t h

H i s 4 3 ,

a s

g e n e r a l l y

o c c u r s

i n

c y s t e i n e

p r o t e a s e s

t o

c l e a v e p e p t i d e - b o n d s .

W e

t h a n k

D r .

M i n g - Y i

L i a u

f o r

p r o v i d i n g

u s

w i t h

v e n o m

g l a n d s . W e a l s o

t h a n k

D r .

T u r - F u

H u a n g

f o r

h e l p f u l a d v i c e a n d

D r .

K o n g - B u n g C h o o

f o r

c r i t i c a l

r e a d i n g

o f

t h e

m a n u s c r i p t .

T h i s

w o r k

w a s

s u p p o r t e d

b y g r a n t

N S C 8 2 - 0 4 1 8 - B 0 7 5 - 0 1 6 - B C

f r o m

t h e

N a t i o n a l

S c i e n c e

C o u n c i l

o f t h e

R e p u b l i c

o f C h i n a .

F i g u r e

4

A l i g n e d a m i n o - a c i d

s e q u e n c e s

o f

t h e

a n c r o d p r e c u r s o r ,

b a t r o x o b i n

p r e c u r s o r ,

f l a v o x o b i n ,

b o v i n e

t h r o m b i n ,

b o v i n e

t r y p s i n a n d h u m a n

k a l l i k r e i n

REFERENCES

T h e

n u m b e r i n g

s t a r t s

f r o m

t h e N - t e r m i n a l r e s i d u e

o f a n c r o d . G a p s

h a v e

b e e n i n t r o d u c e d

t o

o p t i m i z e

t h e

s e q u e n c e

s i m i l a r i t y .

A s t e r i s k s

i n d i c a t e t h e a m i n o - a c i d

r e s i d u e s t h a t a r e

i n v o l v e d

i n t h e

c a t a l y t i c

a c t i o n .

cDNA c l o n e

J - 5

d o e s

e n c o d e

f o r

a n c r o d ,

w h i c h

i s a l s o

a

t h r o m b i n -

l i k e s e r i n e

p r o t e a s e . M o r e o v e r , a 9 3

sequence

i d e n t i t y

b e t w e e n

t h e

f i r s t 3 0 a m i n o - a c i d

r e s i d u e s

o f

t h e

N - t e r m i n i h a s a l l o w e d us

t o

p r e d i c t

t h a t a n c r o d

i s a l s o


as

a

pre-zymogen

b e f o r e

m a t u r a t i o n ,

as

i s

b a t r o x o b i n

a n d

o t h e r s e c r e t o r y s e r i n e

p r o t e a s e s [ 8 , 2 2 - 2 4 ] .

T h e

precursor p e p t i d e i n c l u d e s

a

s i g n a l

p e p t i d e ( p r e - p e p t i d e )

a n d a

z y m o g e n

p e p t i d e

( p r o - p e p t i d e ) .

I t i s

known

t h a t

s i g n a l p e p t i d e s

g e n e r a l l y

c o n t a i n

a

r e g i o n

t h a t i s r i c h

i n

h y d r o p h o b i c a m i n o - a c i d

r e s i d u e s

w i t h

l a r g e

s i d e - c h a i n s ,

a n d

t e r m i n a t e

i n

a

r e s i d u e

h a v i n g

a

s m a l l

s i d e - c h a i n

( A l a ,

S e r ,

or

G l y )

[ 2 5 ] . T h e r e f o r e ,

we

p r e d i c t

t h a t

a

p o s s i b l e

s i t e f o r

t h e

c l e a v a g e

o f

t h e

s i g n a l p e p t i d e

i n t h e

precursor

i s

p r e s e n t

a f t e r t h e A l a r e s i d u e

a t

p o s i t i o n

-7

( t h a t

i s ,

t h e

s i g n a l

p e p t i d e

covers t h e

sequence

f r o m

a m i n o

a c i d s

-2 4 t o

- 7 ; F i g u r e

3 ) .

The

z y m o g e n p e p t i d e

i s

known

t o c o n t a i n

a

r e g i o n

t h a t i s

r i c h

i n

h y d r o p h i l i c

a m i n o -

a c i d r e s i d u e s

[ 8 , 2 2 , 2 3 ]

a n d t h e

f i r s t

N - t e r m i n a l a m i n o a c i d o f

1

O u y a n g , C . , T e n g ,

C . M . a n d

H u a n g ,

T . F .

( 1 9 8 7 )

A s i a P a c .

J . P h a r m a c o l .

2 ,

1 6 9 - 1 7 9

2

E s n o u f ,

M . P .

a n d

T u n n a h ,

G . W .

( 1 9 6 7 )

B r . J .

H a e m a t o l .

1 3 , 5 8 1 - 5 9 0

3

P i z z o ,

S .

V . ,

S c h w a r t z ,

M .

L . ,

H i l l ,

R .

L . a n d

M c k e e ,

P .

( 1 9 7 2 )

J . B i o l . C h e m .

2 4 7 ,

6 3 7 - 6 4 5

4

O u y a n g , C . ,

H w a n g , L .

J . a n d

H u a n g ,

T .

F .

( 1 9 8 3 )

T o x i c o n

2 1 ,

2 5 - 3 3

5

O u y a n g , C . ,

H w a n g , L .

J .

a n d

H u a n g ,

T .

F .

( 1 9 8 5 )

T a i - W a n

H s u c h

H u i

T s a

C h i h

84 ,

1 1 9 6 - 1 2 0 6

6

O u y a n g ,

C . , Y e h ,

H .

a n d

H u a n g ,

T .

F .

( 1 9 8 6 )

T o x i c o n

2 4 ,

6 3 3 - 6 4 3

7

H u a n g ,

T .

F . ,

W u ,

Y .

I.

a n d

O u y a n g ,

C .

( 1 9 8 7 )

B i o c h i m .

B i o p h y s . A c t a

9 2 5 ,

2 4 8 - 2 5 7

8

I t o h , N . , T a n a k a , N . , M i h a s h i ,

S . a n d Y a m a s h i n a I .

( 1 9 8 7 )

J .

B i o l .

C h e m .

2 6 2 ,

3 1 3 2 - 3 1 3 5

9

S h i e h ,

T .

C . , K a w a b a t a ,

S .

I

K i h a r a , K . , O h n o ,

M .

a n d

I w a n a g a , S .

( 1 9 8 8 )

J . B i o c h e m .

( T o k y o ) 1 0 3 ,

5 9 6 - 6 0 5

1 0

P i r k l e ,

H . , T h e o d o r ,

a n d

L o p e z ,

R .

( 1 9 8 9 )

T h r o m b . R e s .

5 6 ,

1 5 9 - 1 6 8

1 1

I t o h , N . , T a n a k a , N . ,

F u n a k o s h i , I K a w a s a k i ,

T . ,

M i h a s h i ,

S . a n d

Y a m a s h i n a ,

I.

( 1 9 8 8 )

J . B i o l .

C h e m .

2 6 3 ,

7 6 2 8 - 7 6 3 1

1 2

S t o c k e r ,

K .

( 1 9 7 8 )

H a n d b . E x p . P h a r m a c o l . 4 6 , 4 5 1 - 4 8 4

1 3

E w a r t ,

M .

R . ,

H a t t o n ,

M . W .

C . , B a s f o r d ,

J . M .

a n d

D o d g s o n ,

K .

S .

( 1 9 7 0 )

B i o c h e m .

J .

1 1 8 ,

6 0 3 - 6 0 9

1 4

L a t a l l o ,

Z .

S .

( 1 9 8 3 )

T h r o m b .

H a e m o s t a s i s

5 0 ,

6 0 4 - 6 0 9

1 5

A u ,

L .

C . , H u a n g ,

Y .

B . ,

H u a n g ,

T .

F . ,

T e h ,

G .

W . , L i n ,

H . H . a n d

C h o o ,

K .

B .

( 1 9 9 1 )

B i o c h e m .

B i o p h y s .

R e s .

C o m m u n .

1 8 1 ,

5 8 5 - 5 9 3

1 6 G r e e n e r ,

A .

( 1 9 9 0 ) S t r a t e g i e s

i n

M o l e c u l a r

B i o l o g y 3 ,

5 - 6

1 7

R u s s e l , M . , K i d d ,

S .

a n d

K e l l y ,

M .

R .

( 1 9 8 6 )

G e n e

4 5 ,

3 3 3 - 3 3 8

3 8 9

A n c r o d

p r e - p r o - p e p

B a t r o x o b i n

p r e - p r o -

A n c r o d

B a t r o x o b i n

F l a v o x o b i n

B o v i n e t h r o m b i n

B o v i n e

t r y p s i n

Human k a l l i k r e i n

A n c r o d

B a t r o x o b i n

F l a v o x o b i n


B o v i n e

t r y p s i n

Human

k a l l i k r e i n

A n c r o d

B a t r o x o b i n

F l a v o x o b i n


B o v i n e

t r y p s i n


A n c r o d

B a t r o x o b i n

F l a v o x o b i n

B o v i ne t h r o m b i n

B o v i n e

t r y p s i n

Human

k a l l i k r e i n

A n c r o d

B a t r o x o b i n

F l a v o x o b i n


B o v i n e

t r y p s i n

Human

k a l l i k r e i n

A n c r o d

B a t r o x o b i n

F l a v o x o b i n


B o v i n e

t r y p s i n

Human

k a l l i k r e i n

A n c r o d

B a t r o x o b i n

F l a v o x o b i n


B o v i n e

t r y p s i n


A n c r o d

B a t r o x o b i n

F l a v o x o b i n


B o v i n e

t r y p s i n

Human

k a l l i k r e i n

A n c r o d

B a t r o x o b i n

F l a v o x o b i n


B o v i n e

t r y p s i n



4/4

3 9 0

L . - C .

A u

a n d o t h e r s

1 8

S a m b r o o k ,

J . ,

F r i t s c h , E .

F . , M a n i a t i s ,

T .

( 1 9 8 9 )

i n

M o l e c u l a r C l o n i n g ,

A

L a b o r a t o r y

M a n u a l ,

2 n d

e d n . , c h a p t e r 1 , C o l d S p r i n g

H a r b o r

L a b o r a t o r y ,

C o l d S p r i n g H a r b o r ,

N e w

Y o r k

1 9

M a x a m ,

A .

M . a n d G i l b e r t ,

W .

( 1 9 8 0 ) M e t h o d s E n z y m o l . 6 5 , 5 0 7 - 5 2 1

2 0

H a t t o n ,

M . W . C .

( 1 9 7 3 )

B i o c h e m . J .

1 3 1 , 7 9 9 - 8 0 7

2 1

P e a r s o n ,

W .

R . a n d

L i p m a n ,

D .

J .

( 1 9 8 8 )

P r o c . N a t l .

A c a d . S c i . U . S . A . 8 5 ,

2 4 4 4 - 2 4 4 8

2 2

M a c D o n a l d , R .

J . , S t a r y ,

S . J .

a n d

S w i f t ,

G . H .

( 1 9 8 2 ) J . B i o l .

C h e m .

2 5 7 ,

9 7 2 4 - 9 7 3 2

2 3 S w i f t , G . H . ,

D a r g o n , J .

C . ,

A s h l e y ,

P .

L . ,

S c o t t ,

W . , C u m m i n g s ,

S .

W .

a n d

M a c D o n a l d ,

R .

J .

( 1 9 8 2 )

P r o c .

N a t l .

A c a d . S c i . U . S . A .

7 9 ,

7 2 6 3 - 7 2 6 7

2 4

D e g e n ,

S .

J .

F . , M a c G i l l i v a r y ,

R .

T .

A . a n d

D a v i e ,

E . W .

( 1 9 8 3 )

B i o c h e m i s t r y

2 2 ,

2 0 8 7 - 2 0 9 7

2 5

S t e i n e r ,

D .

F . , Q u i n n ,

P .

S . ,

C h a n ,

S .

J . ,

M a r s h , J .

a n d

T a g e r ,

H .

( 1 9 8 0 ) A n n .

N . Y .

A c a d . S c i .

3 4 3 , 1 - 1 6

2 6 T a k i o ,

K . ,

T o w a t a r i ,

T . , K a t u n u m a , N . , T e l l e r ,

D . C .

a n d T i t a n i ,

K .

( 1 9 8 3 )

P r o c .

N a t l .

A c a d .

S c i .

U . S . A .

8 0 ,

3 6 6 6 - 3 6 7 0

R e c e i v e d 2 1 J a n u a r y 1 9 9 3 / 1 5 M a r c h 1 9 9 3 ; a c c e p t e d 2 2

M a r c h

1 9 9 3

Documents

1993 Au Molecular Cloning and Sequence Analysis of the CDNA for Ancrod C. Rhodostoma