1997-KHA3A ZAFARULLAH-Synthesis and Conformational Properties.pdf

8/10/2019 1997-KHA3A ZAFARULLAH-Synthesis and Conformational Properties.pdf

1/9

~ ~ ,'~-~

Synthes is and Co nform at iona l Proper ties

o f a R ecom bi nan t C Propept ide o f H um an

Proco[[agen

T y p e

K H A 3A ZA FA R U LLA H E LE A N O R M . B R O WN H E LE N A K U I V A N I E M F G E R A RD

TROMP2 ALEKSANDER L. S IERO N3 A N D R ZE 3 FE RTA LA and D A R W I N 3 . P R O C K O P

Department of Biochemistry and M olecular Biology, efferson Institute of Molecular M edicine, Jefferson

Med ical College, Thomas Jefferson University, Philadelphia, USA

A b s t r a c t

A c D N A w a s p re pa re d th a t c ode d fo r t he s igna l pe p t ide o f t ype II I p roc o l l a ge n l i nke d to

the c omple t e C -p ro pe p t ide o f t he p ro t e in . The c D N A w a s the n u se d to e xp re ss t he p ro t e in i n

a b a c u l o v i r u s r e c o m b i n a n t s y s t e m . R e c o m b i n a n t p r o t e i n w a s r e c o v e r e d a s a t r im e r f r o m t h e

me d ium o f t r a ns fe c t e d c el ls in a y i e ld o f i t o 2 .5 m g pe r l it er . Ma p p ing o f pe p t ide f r a gme n t s

w i t h a n d w i t h o u t r e d u c t i o n i n d i c a t e d t h a t t h e p r o t e i n c o n t a i n e d t h e e x p e c t e d i n t e r c h a i n

d i su l f ide bonds . A na lys i s by c i r c u l a r d i c h ro i sm sugge s t e d tha t t he c on fo rma t ion o f t he p ro -

t e in c o r r e s p o n d e d t o t h e n a t i v e c o n f o r m a t i o n . T h e r e f o r e , t h e p r o t e in s h o u l d b e a p p r o p r i a t e

fo r fu r the r t e s ts o f i ts b io log ic a l func t ion a nd a na ly s i s o f s t ruc tu re by X - ra y d i f f r a c t ion .

K e y w ords : C -p rope p t ide , ba c u lov i ru s e xp re ss ion sys t e m , c i r c u l a r d i c h ro i sm, c o l l a ge n , e x -

t r a c e l lu l a r ma t r ix

I n t r o d u c t i o n

The mos t a bunda n t c o l l a ge ns a re t he f i b r i l l a r t ype s I ,

I I a nd I I I ( se e P roc kop a nd K iv i r ikko , 1995 ) . The y a re

s imi l a r i n s t ruc tu re a nd func t ion , a nd a re a l so s imi l a r i n

tha t t he y a re f i r s t syn the s i z e d a s p roc o l l a ge ns w i th N

1Present address: United States Department of Agriculture,

Eastern Regional Research Center , 600 E. M ermaid Lane,

Philadelphia, PA.

2 Present address: Center for M olecular Medicine and Genet-

ics, Wayn e State University School of Medicine, Detroit, M I.

3 Present address: C enter f or G ene Therapy, A llegheny Uni-

versity of the Health Sciences, Philadelphia, PA.

Abbreviations use d: BM P-1, bonemorphogenic protein-l;

CD, circular dichroism; PA BA, p-aminobenzam idine dihydro-

chloride acid; SF M , serum-free insect cell culture m edium

M a t r ix B io logy V o l. 16 /1997 , pp . 201 - 209

1997 by G ustav Fischer Verlag

t e r m i n a l a n d C - t e r m i n a l p r o p e p t id e d o m a i n s . I n t h e p r o -

c e ss ing o f t he p roc o l l a ge ns to c o l l a ge ns , t he C -p rope p -

t ide s a re c l e a ve d by a s ing le p roc o l l a ge n C -p ro t e ina se

(H o i ima e t a l . , 1985 ) t ha t ha s r e c e n t ly be e n c lone d a nd

show n to ha ve a s t ruc tu re i de n t i c a l t o t he p ro t e in p re v i -

ous ly de f ine d a s bone morp hoge n ic p ro t e in -1 (B MP-1 )

(Kessle r e t a l. , 1996; L i e t a l. 1996) . T he N -pro pep t ides

o f t ype I a nd type I I p roc o l l a ge n a re c l e a ve d by a spe c i f ic

p roc o l l a ge n N -p ro t e ina se (H o j ima e t a l . , 1989 ) w h i l e

the N -p rope p t ide o f t ype I I I p roc o l l a ge n i s c l e a ve d by a

se pa ra t e N -p ro t e ina se (H a l i l a e t al . 1986 ) . A f t e r cl e a v-

a ge o f t he tw o p rope p t ide s , t he r e su l t i ng c o ll a ge ns spon -

t a ne ous ly se l f - a sse mble in to f i b r i l s ( se e P roc kop a nd

H ulme s , 1994 ) .

The C -p rope p t ide s o f t he f i b r i l l a r p roc o l l a ge ns p l a y a

c r i t ic a l ro l e i n t he b io syn the s i s o f t he p ro t e in s (P roc kop


2/9

2 0 2 K. Za f a r u l l a h e t a l .

a n d K i v i r i k k o , 1 9 9 5 ) . T h e y d i r e c t c h a i n s e l e c t i o n a n d

s t a b i l i z e t h e a s s o c i a t e d c h a i n s b y t h e f o r m a t i o n o f i n t e r -

c h a i n d i s u l f id e b o n d s . T h e y a l s o p r o v i d e r e g i s t r a t io n o f

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

C - t e r m i n a l e n d o f t h e l a rg e c o l la g e n d o m a i n o f t h e p r o -

t e in a n d t h e t r ip l e h e l i x is th e n p r o p a g a t e d f r o m t h e C -

t e r m i n u s t o t h e N- t e r m i n u s ( Sc h o f i e l d e t a l . , 1 9 7 4 ; Ui t t o

a n d P r o c k o p , 1 9 7 4 a , b ; B~ i c hi ng e r e t al . , 1 9 8 0 ) . Th e c r i ti -

c a l r o l e o f t h e C- p r o p e p t i d e s i n t y p e I p r o c o l l a g e n i n d i -

r e c t in g c h a i n a s s o c i a ti o n w a s d e m o n s t r a t e d b o t h b y a s-

s a y s

in vitro

a n d b y n a t u r a l l y o c c u r r i n g m u t a t i o n s i n

m a n o r m i c e t h a t a l t e r th e s t r u c t u r e o f t h e C - p r o p e p t i d e s

a n d t h e r e b y p r e v e n t c o r r e c t c h a i n a s s o c i a t io n ( N i c h o ll s

e t a l . , 1 9 7 9 ; De a k e t a l . , 1 9 8 3 ; Ch i p m a n e t a l . , 1 9 9 3 ;

La m a n d e e t a l . , 1 9 9 5 ; s e e a l s o Ku i v a n i e m i e t a l . , 1 9 9 7 ) .

I n a d d i t i o n , t h e r e a r e s u g g e s t i o n s t h a t t h e C - p r o p e p -

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

s e r v e a s f e e d - b a c k r e g u l a t o r s o f c o l l a g e n b i o s y n t h e s i s e i -

t h e r a t t h e t r a n s c r i p t i o n a l l e v e l ( W u e t a l . , 1 9 8 6 ) o r t h e

p o s t - t r a n s c r i p t i o n a l l e v e l ( Ay c o c k e t a l . , 1 9 8 6 ; Ka t a y -

a m a e t a l. , 1 9 9 1 ; W u e t a l . , 1 9 9 1 ) . A l s o , t h e r e a r e i n d i c a -

t i o n s t h a t C - p r o p e p t i d e s o f t y p e I p r o c o l l a g e n b i n d t o

c e l l s u r f a c e s t h r o u g h t h e 0 t2 13 1 i n t e g r i n a n d t h e r e b y p l a y

a r o l e i n c e l l a t t a c h m e n t a n d s p r e a d i n g ( W e s t o n e t a l . ,

1 9 9 4 ) .

T h e r e i s a h i g h d e g r e e o f a m i n o a c i d h o m o l o g y a m o n g

t h e C - p r o p e p t i d e s o f t y p e I , t y p e I I a n d t y p e I II p r o c o l l a -

g e n s ( D i o n a n d M y e r s , 1 9 8 7 ) . E a c h o f th e p r o p e p t i d e s

h a s e i g h t c y s t e i n e r e s i d u e s a n d a n N- g l y c o s y l a t i o n s i t e

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

e n t c h a i n s . A l s o , t h e p o s i t i o n s o f t h e t y r o s i n e , t r y p t o -

p h a n a n d c h a r g e d r e s i d u e s , a s w e ll a s th e p a t t e r n s o f h y -

d r o p h i l i c i t y a n d h y d r o p h o b i c i t y , a r e c o n s e r v e d ( D i o n

a n d M y e r s , 1 9 8 7 ) . S t u d i e s

in vitro

o n t h e C - p r o p e p t i d e s

o f t y p e I a n d t y p e I I I p r o c o l l a g e n s s u g g e s t e d t h a t c y s -

t e i n e r e s i d u e s a t t h e a m i n o t e r m i n a l r e g i o n f o r m i n t e r -

c h a i n d i s u l fi d e b o n d s , w h e r e a s t h e m o r e C - t e r m i n a l c y s -

t e i n e r e s i d u e s a r e i n v o l v e d i n t h e f o r m a t i o n o f i n t r a -

c h a i n d i s u l f i d e b o n d s ( Ko i v u , 1 9 8 7 ; Le e s a n d Bu l l e i d ,

1 9 9 4 ) .

D e t a i l e d s t r u c t u r a l a n a l y si s o f t h e C - p r o p e p t i d e h a s

b e e n l i m i t e d i n p a r t b y t h e p a u c i t y o f t h e p r o t e i n a v a i l -

a b l e f r o m n a t u r a l s o u r c e s . H e r e , w e e x p r e s s e d a c D N A

f o r t h e C - t e r m i n a l p r o p e p t i d e o f t y p e I II p r o c o l l a g e n i n a

b a c u l o v i r u s e x p r e s s i o n s y s t e m . T h e e x p r e s s e d r e c o m b i -

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

f o r f o r m a t i o n o f th e c o r r e c t d i s u lf i d e b o n d s b y c y a n o g e n

b r o m i d e m a p p i n g o f th e p e p t i d e s u n d e r r e d u c i n g a n d

n o n - r e d u c i n g c o n d i t i o n s . A l s o , t h e c o n f o r m a t i o n o f t h e

p r o t e i n a n d i ts t h e r m a l s t a b il i ty w e r e a s s a y e d b y c i r c u l a r

d i c h r o i s m ( C D ) .

aterials and ethods

Ampli f icat ion and sub-cloning of type III procol lagen

C-propeptide

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

t h e C- p r o p e p t i d e o f t y p e I I I p r o c o l l a g e n we r e a s s e m b l e d

a s t wo f r a g m e n t s b y u s i n g t h e f u l l - le n g t h t y p e I II p r o c o l -

l a g e n c D N A c l o n e ( H . K u i v a n i e m i , G . T r o m p , u n p u b -

l i shed resul t s ) as a t empla te on spec i f i c pr imers in PCR

( Sa i k i e t a l . 1 9 8 5 ) . To a m p l i f y s e q u e n c e s c o d i n g f o r t h e

s i g n a l p e p t i d e , t h e u p s t r e a m p r i m e r wa s KZ- 3 5 - I I I 5 ' -

A A G A T C T A A A G A G T C T C A T G T C T G A T A T T T A -

3 ' t h a t i n i t i a t e d a t b a s e n u m b e r 7 6 f r o m t h e s t a r t o f t r a n -

s c r i p t i o n a n d t h a t c r e a t e d a u n i q u e BgllI s i te on the 5 ' t e r -

m i n i . T h e d o w n s t r e a m p r i m e r w a s K Z - 3 6 - I I I 5 -AAT

C G A T C T G T T G T G C C A A A A T A A T A G T - 3 ' t h a t te r-

m i n a t e d a t b a s e n u m b e r 1 7 7 a n d t h a t c r e a t e d a u n i q u e

Cl a I s i t e o n i t s 5 ' t e r m i n u s . Th e p r i m e r s we r e b a s e d o n

t h e p u b l i s h e d c DNA s e q u e n c e ( Al a - Ko k k o e t a l . , 1 9 8 9 ) .

Th e a m p l i f i c a t i o n c o n d i t i o n s we r e : a n i n i t i a l d e n a t u r a -

t i o n a t 9 4 . 5 C f o r 2 m i n , f o l l o w e d b y 3 0 c y c l e s o f d e n a t -

u r a t i o n a t 9 4 C f o r 1 5 s e c , a n n e a l i n g a t 6 0 C f o r 1 5 s ec

a n d e l o n g a t i o n a t 7 2 C f o r 1 5 s e c , f o l l o we d b y a f i n a l

e l o n g a t i o n a t 7 2 C f o r 7 m i n . T h e P C R p r o d u c t s w e r e

a n a l y z e d o n 0 . 8 % a g a r o s e g e l . c D N A s e q u e n c e s c o d i n g

f o r t h e c o m p l e t e C - p r o p e p t id e a n d a p o r t i o n o f th e 3 ' - u n -

t r a n s l a t e d r e g i o n we r e a m p l i f i e d u s i n g t h e p r i m e r KZ- 3 7 -

Il l 5 -ATT C G A T G A A C C A A T G G A T T T C A A A A T

CAA C- 3 ' t h a t i n i t i a t e d a t p o s i t i o n n u m b e r 3766 f r o m

t h e s t a r t o f t r a n s c r i p t i o n a n d c r e a t e d a u n i q u e ClaI s i t e on

t h e 5 ' - t e r m i n i , a n d t h e p r i m e r K Z q l I - 1 8 5 ' - G G C G A A

G C T T G T T G A C C T T T A C A G G A T G A A - 3 ' th a t te rm i -

n a t e d a t b a s e n u m b e r 5 3 3 7 i n t h e 3 ' - u n t r a n s l a t e d r e g i o n

o f th e c DN A. T h e PC R c o n d i t i o n s u s e d we r e a n i n it i al

d e n a t u r a t i o n a t 9 4 . 5 C f o r 3 0 s e c , f o l l o we d b y 3 0 c y c l e s

o f d e n a t u r a t i o n a t 9 4 C , a n n e a l i n g a t 5 5 C f o r 3 0 s ec ,

a n d e l o n g a t i o n a t 7 2 C f o r 9 0 s e c f o l l o we d b y a f i n a l

e l o n g a t i o n a t 7 2 C f o r 7 m i n . T h e P C R p r o d u c t o f 1 5 8 6

b p c o n t a i n e d a ClaI s i t e e n g i n e e r e d o n KZ- 3 7 - I I I a n d a

n a t u r a l X b a I s i t e i n t e r n a l t o t h e PCR p r i m e r I I I - 1 8 t h a t

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

f o r th e t y p e I II s ig n a l - C O O H p r o p e p t id e . T h e P C R p r o d -

u c t wa s a n a l y z e d o n a 1 % a g a r o s e g e l a n d c l o n e d i n t o t h e

v e c t o r p CRI I ( I n v i t r o g e n ) . I n d i v i d u a l c l o n e s we r e s e -

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

d u r i n g t h e P C R . O n e c l o n e c o n t a in i n g t h e c D N A f o r th e

s i g n a l p e p t i d e a n d a n o t h e r c l o n e c o n t a i n i n g t h e C-

p r o p e p t i d e c D N A f r a g m e n t w e r e e a c h d i g e s t e d w i t h t h e

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

ClaI

( Ne w En g l a n d B i o l a b s ) a n d

l i g a t e d t o g e t h e r i n t o t h e

BgllI

a n d

XbaI

in the vec tor


3/9

pCR II ( Invi t rogen) using T4 DN A l igase (USB) a t 16 C

ove rn igh t (Sa mbrook e t a l . 1989 ) . The re su l t a n t l i ga t ion

p roduc t s w e re a mp l i f i e d u s ing the p r ime rs K Z-35 - I I I a nd

K Z-I I I -18 a nd P C R c on d i t i ons i de n t ic a l t o t hose u se d fo r

the a mp l i f i c a t ion o f t he C -p rope p t ide ( se e a bove ) . The

PC R p roduc t w a s a na lyz e d on a 1% a ga rose ge l , c lone d

into the vec tor pCRII ( Invi t rogen) , and the sequence ver i -

f i ed by D N A se que nc ing .

Construction o f the transfer vector p VL-TyH I-3.1

T h e r e c o m b i n a n t c l o n e th a t c o n t a i n e d b o t h t h e s i gn a l

p e p t i d e a n d C - p r o p e p t id e w a s e x c i s ed w i t h t h e e n z y m e s

BglI

(N e w Eng la nd B io l a bs ) a nd

XbaI

(N e w Eng la nd B i -

o l a bs ) , a nd subc lone d in to t he sa me s i t e s i n t he ba c -

u lov i ru s t r a ns fe r ve c to r pV L1392 ( Inv i t roge n ) . R e s t r i c -

t i o n e n z y m e d i g e s t i o n a n d D N A s e q u e n c i n g w e r e u s e d

to ve r i fy the o r i e n t a t ion in t he shu t t l e ve c to r o f t he i n -

se r t s o f pos i t i ve c lone s w i th r e spe c t t o t he po lyhe d ron

(po lh ) p ro mo te r (O 'R e i l l y e t a l. 1992 ) .

Co-transfection of transfer vector and viral DN A

Spodoptera frugiperda

(Sf9) ce l ls (Brown and Faulkner

1977) (3 x 106 ce l ls per 25 cm 2 t i ssue cul tu re f lask) w ere

c o - t r a ns fe c t e d w i th pV L-TyI I I -3 .1 a nd Autographica cal-

ifornica

nuc le a r po lyhe d ros i s v i ru s (A c N PV ) D N A (B a c -

u logoldTM; Pha rminge n ) u s ing t r a ns fe c t ion bu f fe r a s

spec i f ied by the suppl ie r . The t ransfec ted ce l ls were incu-

ba t e d in G ra c e ' s (non -supp le me n te d ) i n se c t c e l l c u l tu re

me d ium p lus 10% fe t a l bov ine se rum a t 27 C fo r 6 da ys

be fo re t he c e l l c u l tu re supe rna t a n t c on ta in ing the e x t ra -

c e l lu l a r r e c ombina n t ba c u lov i ru s ( rB a c -Ty l I I -3 .1 ) w a s

col lec ted and used to infec t f resh Sf9 ce l ls for v i rus am-

pl i f ica t ion . High t i te r v i rus s tocks of rBac-TylII-3 .1 (4 x

108 v iru s pa r t ic l e s /ml ) w e re o b ta ine d a f t e r tw o round s o f

a mpl i f i c a t ion (P2 a nd P3 v i ru s ) i n w h ic h the r e c om bina n t

ba c u lov i ru s - in fe c t e d c e l l s w a s inc uba te d a t 27 C fo r 4

days each in Sf900II se rum-free insec t ce l l cul ture

me d ium (SFM; G ibc o B R L Li fe Te c hno log ie s , Inc . ).

Expression of TylII-3.1 recombinant protein in insect

cells

Trichoplusia ni

(5B1-4, High Five) cells (1 x 106 per

ml ) ( Inv i t roge n C orp . ) w e re i n fe c t e d w i th rB a c -TyII I -3 .1

a t a mu l t i p l i c i t y o f i n fe c t ion o f a pp rox ima te ly 5 v i ru s

pa r t ic l e s /c e l l a nd inc uba te d in S f900 I I SFM. The c e ll c u l-

t u r e s u p e r n a t a n t s c o n t a i n i n g t h e T y I I I - 3 . 1 r e c o m b i n a n t

p ro t e in w e re a na lyz e d by e l e c t ropho re s i s on 4 to 1 5%

SD S -po lya c ry l a mide gel s (B ioR a d) a nd the p ro t e in

R e c o mb ina n t C -Prope p t ide I I I 203

b a n d s s t a i n e d w i t h C o o m a s s i e b l ue ( R 2 5 0 ; K o d a k ) u s i n g

s t a n d a r d m e t h o d s ( S a m b r o o k e t a l. 1 9 8 9 ) .

Scale-up production of Ty III-3.1

La rge qua n t i t i e s o f Ty I I I -3 .1 r e c ombina n t p ro t e in

w e re p rodu c e d in H igh F ive C el ls by in fe c t ing 1 L o f su s-

pension cul ture (1 106 ce l ls per ml) wi th rBac-TyIII-3 .1

v i ru s i n a 3 l i t e r sp inne r f l a sk f i t te d w i th a doub le pa dd le

(Bel lco Glass, Inc .) . When the ce l l v iabi l i ty reached 90%

(48 h p . i .) , t he ce l l c u l tu re sup e rna t a n t w a s ha rve s t e d by

c e n t r i fuga t ion in 250 ml bo t t l e s a t 5000 g fo r 10 min ,

a nd t r a ns fe r re d to f r e sh bo t t l e s .

Purification of C- propeptide from media

p - A m i n o b e n z a m i d i n e d i h y d r o c h l o r i d e a c i d ( P A B A ;

S i g m a ) t o a f i n a l c o n c e n t r a t i o n o f 1 m M , a n d N - e t h y l -

ma le imide (N EM; S igma ) to a f i na l c onc e n t ra t i on o f

1 0 m M w e r e a d d e d t o t h e ce ll c u lt u r e s u p e r n a t a n t c o n -

t a in ing the r e c ombina n t t ype I I I p roc o l l a ge n C - p rope p -

t ide . The sa mple w a s f i l t e re d to r e move c e l l de b r i s a nd

pa sse d ove r a C on A Se pha rose 4B ma t r ix (Pha rma c ia )

a s de sc r ibe d by C hog la y et a l. (1993 ) . B o und p ro t e in s

w e re e lu t e d w i th M m e t h y l - ~ - D - m a n n o - p y r a n o s i d e

a nd f r a c t ions c o l l e c t e d . Sa mple s f rom the f r a c t ions w e re

se pa ra t e d by SD S-PA G E unde r bo th r e duc ing a nd non -

re duc ing c ond i t i ons a nd p roduc t s v i sua l i z e d by s t a in ing

w i th C ooma ss i e B r i l l i a n t B lue . A l l f r a c t ions t ha t c on -

t a ine d p ro t e in w e re poo le d a n d d i a lyz e d tw ic e a ga in s t

10 vo lume s o f c a t ion bu f fe r (Pha rma c ia ) c on ta in in g

5 0 m M s o d i u m ac e ta t e (p H 4 . 5) , 5 m M N E M a n d

0 . 5 m M P A BA . T h e p r o t e i n s w e r e l o a d e d o n t o 2 5 m l o f

c a t ion bu f fe r e qu i l i b ra t e d SP Se pha rose Fa s t F low c a t ion

m a t r i x ( P h a r m a c ia ) a t a f l o w r a t e o f 5 0 - 7 5 m l / h . T h e

c o l u m n w a s w a s h e d w i t h 5 - 1 0 c o l u m n v o l u m e s o f

c a t ion bu f fe r a nd the p ro t e in e lu t e d w i th a n N a C I g ra -

dient , 0 to 1 M in one l i te r . Five mil l i l i te r f rac t ions were

c o l l e c t e d a nd a bso rb a nc e a t 28 0 }am me a su re d . The C -

p rope p t ide e lu t e d a s a s ing le pe a k . A sa mple o f 100 -500

ng o f t he p ro t e in w a s se pa ra t e d by e l e c t ropho re s i s on a

4 to 15 % SD S-po lya c ry l a mide ge l (B ioR a d) a nd v i sua l-

i z e d by s t a in ing w i th C o oma ss i e b lue .

C - p rope p t ide o f t ype I p roc o l l a ge n w a s pu r i f i e d f rom

c h ic ke n t e ndon c u l tu re s a s de sc r ibe d by C hog la y e t a l .

(1993) .

Generation o f cyanogen bromide fragments

Pur i f i e d C -p rope p t ide s i n 70% fo rmic a c id w e re r e -

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


4/9

2 0 4 K. Za f a r u l l a h e t a l .

1 0 0 ~ 1 o f p r o t e i n s o l u t i o n ( a b o u t 1 m g ) a t r o o m t e m p e r -

a t u r e f o r 1 4 - 1 8 h . T h e s a m p l e w a s l y o p h i l i z e d a n d r e -

s u s p e n d e d i n s t o r a g e b u f f e r ( 0 . 1 M Tr i s - HCl , 0 . 4 M

N a C I , a n d 2 5 m M E D T A , p H 7 . 4 ) a n d s e p a r a t e d b y

e l e c t r o p h o r e s i s t h r o u g h a 4 - 1 5 % g r a d i e n t p o l y a c r y -

l a m i d e g e l i n S D S u n d e r e i t h e r r e d u c i n g o r n o n - r e d u c i n g

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

Co o m a s s i e b l u e o r s i l v e r s t a i n i n g o f t h e g e l.

CD spec t roscopic ana lys i s

C i r c u l a r d i c h r o i s m ( C D ) s p e c t r a w e r e r e c o r d e d o n a n

A V T V 6 0 D S s p e c t r o m e t e r ( A V I V A s s o c i a t e s , L a k e w o o d ,

N J ) e q u i p p e d w i t h a t h e r m o s t a t e d c e l l h o l d e r c o n n e c t e d

t o a p r o g r a m m a b l e c i r c u l a t i n g w a t e r b a t h ( L a u d a M o d e l

R C 6 f r o m B r i n k m a n n I n s t ru m e n t s , W e s t bu r y , N Y ) . T h e

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

( C h e n a n d Y a n g , 1 9 7 7 ) . S a m p l e c o n c e n t r a t i o n s w e r e

7 . 4 x 1 0 - s g / m l f o r t h e c h i c k e n t y p e I C- t e r m i n a l p r o p e p -

t i d e a n d 5 , 7 x 1 0 - s g / m l f o r t h e h u m a n t y p e I l l C- t e r m i -

n a l p r o p e p t i d e a s d e t e r m i n e d u s i n g t h e B r a d f o r d ( 1 9 7 6 )

a s s a y ( B i o R a d , H e r c u l e s , C A ) . T h e s a m p l e s w e r e d i a -

l y z e d i n t o 1 0 m M T r i s - H C l b u f f e r a n d p l a c e d in 0 .1 c m

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

( H e l l m a , J a m a i c a , N Y ) . A f t er t h e s a m p l e w a s e q u i li -

b r a t e d i n t h e i n s t r u m e n t a t 2 0 C f o r 3 0 m i n , t h e f a r U V

C D s p e c t ru m f r o m 2 5 0 n m t o 1 9 5 n m w a s r e c o r d e d . T o

a s s a y t h e r m a l s t a b i l i t y , t h e t e m p e r a t u r e wa s i n c r e a s e d a t

a r a t e o f 0 . 3 C / m i n a n d t h e e l l i p t i c i t y a t 2 2 0 n m wa s

r e c o r d e d e v e r y 5 C b e t w e e n 2 0 C a n d 6 0 C . A t 6 0 C

a f i n al s p e c t r u m w a s r e c o r d e d .

R e s u ~ s

T ime course o f pro te in induc t ion

T h e r e c o m b i n a n t v i r u s , r B a c - T y I I I - 3 . 3 c o n t a i n e d t h e

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

t o t h e C - p r o p e p t i d e o f t y p e I I I p r o c o l l a g e n . T h e f u s e d

f r a g m e n t s c o n t a i n e d a n e w ClaI s i t e a n d a n a d d i t i o n a l

c o d o n f o r I l e a t t h e C- t e r m i n u s o f t h e s i g n a l s e q u e n c e .

Ba c u l o v i r u s c e l l s we r e i n f e c t e d wi t h r e c o m b i n a n t v i r u s

r Ba c - Ty I I I - 3 . 1 a n d t h e m e d i a wa s r e m o v e d f r o m t h e c e l l

c u l t u r e a f t e r 2 4 , 4 2 , 4 8 , 6 5 a n d 7 2 h p o s t - i n f e c t i o n . Th e

p r o t e i n s we r e s e p a r a t e d o n SD 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 u n d e r b o t h r e d u c e d a n d n o n r e d u c e d c o n d i -

t i o n s . A h i g h l y i n d u c e d b a n d wa s i n i t i a l l y s e e n i n t h e

m e d i a a f t e r 2 4 h a n d 4 2 h i n f e c t i o n ( F i g . 1 ) . Th e t o t a l

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

6 5 a n d 7 2 h . T h e r e f o r e , t h e re w a s n o e v i d e n c e o f m a r k e d

i n t r a c e l l u l a r a c c u m u l a t i o n o f t h e p r o t e i n . As e x p e c t e d

f o r t h e C- p r o p e p t i d e o f t y p e I I I p r o c o l l a g e n , t h e r e c o m b i -

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

w i t h a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 1 0 0 k D a ( n o t

s h o w n ) . A f t e r r e d u c t i o n , i t w a s a m o n o m e r o f a p p r o x i -

9 7 . 4

6 6 . 2

4 5 . 0

3 1 . 0

2 1 . 5

1 4 . 4

6 . 5

1 2 3 4 5 6 7 M

Figure 1. Time course o f recom bina nt C-propeptide of proa~xl(III) collagen expression using a baculovirus system. Sam ples of

medium were reduced an d electrophoresed throug h a 4- 15 % SDS-polyacrylamide gel and proteins stained using Coomassie Blue.

Lanes 1 and 7, total protein from uninfected cel ls. Lanes 2-6 , total protein 24, 42, 48, 65, and 72 h post- infect ion. A rrow indicates

expressed C-propeptide. M, molecular weight markers (BioRad Broad Range) with values in kDa.


5/9

m a t e l y 3 2 k D . A f t e r i n c u b a t i o n o f th e c e ll s f o r 6 5 a n d 7 2

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

a b o u t 3 0 k D a u n d e r r e d u c i n g c o n d i t i o n s , a n o b s e r v a t i o n

s u g g e s ti n g d e g r a d a t i o n o f t h e r e c o m b i n a n t p r o t e i n a t t h e

l a t e r ti m e p o i n t s ( F ig . 1 ) . Ad d i t i o n o f p r o t e a s e i n h i b i t o r s

d u r i n g c e l l c u l t u r e h a d l i tt l e e ff e c t i n p r e v e n t i n g d e g r a d a -

t i o n o f t h e r e c o m b i n a n t p r o t e i n .

Purification o f reco mb inant material from insect cell

culture

R e c o m b i n a n t C - p r o p e p t i d e o f t y p e I I I p r o c o l l a g e n s e -

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

m a t o g r a p h y o n a c o n c a n a v a l i n A c o l u m n a n d t h e n o n a

c a t i o n e x c h a n g e c o l u m n . T h e b o u n d p r o t e i n w a s e l u t e d

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

a n d t h e C - p r o p e p t i d e o f p r o i x l( I I I) c o l l a g e n e lu t e d a t

0 . 1 5 M N a C I . T h e f r a c t io n s w e r e a n a l y z e d b y SD 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 ( Fi g. 1 ). Th e t y p i c a l y i e ld s

o f p u r i f ie d p r o t e i n w e r e 1 t o 2 . 5 m g p e r l it e r o f m e d i a .

Characterization o f inter - and intra-chain disulfide

bonds by cyanogen b romide cleavage

T h e r e c o m b i n a n t p r o t e i n w a s a s s a y e d f o r p r e s e n c e o f

d i s u lf i d e b o n d s b y c y a n o g e n b r o m i d e m a p p i n g (F ig . 3 ).

Th e c y s t e i n e s n u m b e r e d C I t o Ca t h a t l ie c l o s e s t t o t h e

t r i p l e h e l ix o f t h e m o l e c u l e , h a v e b e e n s h o w n t o b e in -

v o l v e d i n i n t e r - c h a i n d i s u l f i d e l i n k a g e s ( Ko i v u , 1 9 8 7 ;

L e e s a n d B u l l e i d , 1 9 9 4 ) . I n u n r e d u c e d c o n d i t i o n s t h e y

s h o u l d g e n e r a t e a d i s u lf i d e -l i n k e d f r a g m e n t o f 2 0 . 5 k D a .

T h e C - t e r m i n a l c y s t e i n e s C s t o C 8 f o r m i n t r a - c h a i n

d i s u l f i d e l i n k a g e s . U n d e r n o n - r e d u c i n g c o n d i t i o n s t h e y

s h o u l d g e n e r a t e a f r a g m e n t o f 1 7 . 4 k D a . T h e r e c o m b i -

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

s e p a r a t e d b y S D S - P A G E u n d e r n o n r e d u c i n g o r r e d u c i n g

c o n d i t i o n s . A s e x p e c t e d , t h e l a r g e s t p e p t i d e s o b t a i n e d

u n d e r n o n - r e d u c i n g c o n d i t i o n s w e r e a b o u t 2 0 k D a a n d

1 7 k D a ( a r r o w s A a n d B i n Fig . 3 B ). T h e m o l e c u l a r

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

r e l a te t o t h e e x p e c t e d s i ze s a p p a r e n t l y b e c a u s e o f i n c o m -

p l e te c l e a v a g e b y c y a n o g e n b r o m i d e .

R e c o m b i n a n t C - P r o p e p t i d e I II 2 0 5

1 7 5 0 - -

8 3 . 0

6 2 . 0

4 7 . 5 - -

3 2 5 - -

2 5 . 0 - -

1 6 . 5 - -

M 1 2 3 4 5

1 7 5 . 0

8 3 . 0

6 2 0

4 7 . 5

3 2 . 5

2 5 . 0

16 .5

Figure 2. Purif icat ion of recombinant C-propept ide of

proixl(III) collagen fro m insect cell cultures. S amples were elec-

trophoresed through a 4-15% SDS-polyacrylamide gel and

proteins stained using Coomassie B lue. Panel A, reducing con-

ditions; Panel B, non-reducing conditions. Lane 1, media from

uninfected cel ls as negat ive control ; Lane 2, total media 48 h

post-infections; Lane 3, void volume from concanavalin col-

umn; Lane 4, bo und proteins from concanaval in colum n eluted

with methyl-a-D-mannopyranoside; Lane 5, bound protein

eluted with salt gradient from SP Sepharose cation column. M,

molecular weight marker (New England Biolabs) with values

in kDa. Arrow indicates expressed C-propeptide.

Analysis o f protein structure by circular dichroism

spectroscopy

T h e s p e c t r u m s h o w e d a b r o a d p e a k o f n e g at i v e el li p -

t i ci t y i n t h e w a v e - l e n g t h r a n g e o f 2 0 0 t o 2 4 0 n m f o r

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

( Fi g. 4 A) . An a l y s i s o f t h e s p e c t r u m b y th e a l g o r i t h m o f

Ch e n e t a l . ( 1 9 7 2 ) i n d i c a t e d t h a t t h e t r i m e r c o n s i s t s o f

Table I . CD spectra of hum an reco mbinan t type III and chick

type I C-propeptides.

a - he li x i x - s h e e t I x - t u r n R a n d o m

Hu m a n t y p e I II 7 % 3 1 . 7 % 2 7 . 8 % 3 3 . 3 %

Chick type I 7 .7% 39.8% 20.8% 31.4%

Values from Spectra (Fig. 4A) were normalized to 100%.


6/9

2 0 6 K . Z a f a r u l l a h e t a l .

Amino Acid ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

~ ~ ~ ~

I I I I I I I

C a l c u l a t e d M W

K O ) ~ ~ 1 6 . ~ 4 . 0 2 ~ .3 1 ~ . s

M I 9

Cv

U

8 . t

o ,

I

B

2 1 2 . 0 - -

1 7 0 . 0 - -

1 1 6 . 0 _ _

7 6 . 0 - -

5 3 . 0 - -

r ~ ~--

H M W M 1 2 3 4 L M W M

- - 1 6 . 9

- - 1 4 . 4

- - 1 0 . 7

- - 8 1 5

6 . 2 1

Figure 3. A. Diagram ma tic illustration of expected sizes of fragmen ts after cyanoge n bromide cleavage. Top panel represents a sin-

gle chain of the C-prope ptide from proct(III) collagen. Nu mb ers at top repre sent amino acid positions; numb ers at the botto m rep-

resent calculated molecular weights of each fr agm ent after cleavage with cy anogen brom ide; Ml-s represents m ethionine residues;

C~_8 rep res en t cysteine residue s.

B. Inter- and intra-chain disulfide bond characterization by cyanogen bromide cleavage. Lane HMWM: High molecular weight

markers (Pharmacia). Lane 1: Unreduced recombinant C-propeptide. Lane 2: Reduced recombinant C-propeptide. Lane 3:

Cyano gen bromide fragm ents of C-prope ptide, unreduced. Lane 4: Cyanogen bromide digest of C-propep tide, reduced. Lane

LMWM: Low molecular weight markers (Pharmacia) in kDa. The arrows A and B indicate fragments with intrachain and inter-

chain disulfide bonds, respectively.

7 . 0 % ~ - h e l i x , 3 1 . 7 % ~ - s h e e t, 2 7 . 8 % 1 3- tu rn a n d 3 3 . 3 %

r a n d o m s t r u c t u r e ( T a b l e 1 ) . S i n c e t h e a m i n o a c i d c o m -

p o s i ti o n o f t he h u m a n t y p e I I I C - p r o p e p t i d e ( A l a - K o k k o

e t a l 1 9 8 9 ) i s h i g h l y h o m o l o g o u s t o t h e c h i c k e n t y p e I

c o l l a g e n C - p r o p e p t i d e ( B e r n a r d e t a l . 1 9 8 3 a , b ) , t h e s e c -

o n d a r y s t r u c t u r e o f t h e C - p r o p e p t i d e o f c h i c k e n t y p e I

p r o c o l la g e n o b t a i n e d f r o m o r g a n c u l tu r e s o f t e n d o n s

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

t h e t r i m e r o f t y p e I p r o c o l l a g e n C - p r o p e p t i d e s h a d

a b o u t t h e s a m e s t r u c t u r e a s t h e r e c o m b i n a n t t y p e I II C -

p r o p e p t i d e ( T a b l e 1 ).

T h e C D s p e c t r a o b t a i n e d a t 2 0 C a n d a t 6 0 C f o r t h e

t w o C - p r o p e p t i d e s w e r e q u a l i t a t i v e l y s im i l a r. T h e r e w a s

a s l i g h t d ec rease i n t h e r e l a t i v e am o u n t o f c~ -h e l ix a t

6 0 C a c c o m p a n i e d b y a c o r r e s p o n d i n g i n c r e a s e i n u n -

o r d e r e d s t r u c t u r e s ( n o t s h o w n ) . T h e r e s u l t s , h o w e v e r ,

w ere w e l l w i th in t h e -+ 5 % l im i t s o f t h e m e th o d . A s a

m o r e d i r e c t m e a s u r e o f c o n f o r m a t i o n a l s t a b il i ty , t h e

d a t a i n F i g u r e 4B w e r e o b t a i n e d b y m o n i t o r i n g t h e e l li p -

t i c it y a t 2 2 0 n m o n a s i n g le s a m p l e o f e a c h p r o p e p t i d e

a s t h e t e m p e r a t u r e w a s s l o w l y i n c r e a s e d f r o m 2 0 C t o

6 0 C . T h i s p l o t i n d i c a t e d t h a t t h e h u m a n r e c o m b i n a n t

t y p e I I I C - p r o p e p t i d e w a s m o r e s t a b l e t h a n t h e c h i c k

t y p e I C - p r o p e p t i d e . C o m p a r i s o n s o f t h e fu l l s p e c t r a a t

6 0 C ( n o t s h o w n ) w i t h t h e 2 0 C s p e c t r a s u p p o r t e d t h e

s a m e c o n c l u s i o n .


7/9

l ]

5OO

-5OO

-1000

-1500

-2000

2OO

i i

- - - - - H U M A N T Y P E I I I

/

/

la

/

210

2 2 0 2 3 0

W o v e l e n g t h n m )

Recom binant C -Propept ide I II

2 4 0 2 5 0

2 0 7

[ e ] . ~ .

-9OO

-1000

-1100

-1200

-1300

-1400

o D

_ m S

o

I C H I C K E N T Y P E I

- D . H U M A N T Y P E I I I

I , I ~ I I , I

2 0 3 0 4 0 5 0 6 0

Tomperaturo ( C)

Figure 4. CD spectroscopic analysis of C-propeptide of chicken type I and human r ecombi nant type III procollagens. A CD spec-

trum of the propeptides a t 20 C. The data are expressed in terms of the mean residue eUipticity [0]. B A plot of [01220nm as a

function of temperature between 20 C and 60 C.


8/9

2 0 8 K . Z a f a ru l l ah e t a l .

iscussion

T h e r e s u l t s h e r e d e m o n s t r a t e t h a t t h e C - p r o p e p t i d e o f

h u m a n t y p e

I I I

p r o c o l l a g e n c a n b e s y n t h e s i z e d i n a b a c -

u l o v i r u s r e c o m b i n a n t s y s t e m . T h e p r o t e i n w a s s e c r e t e d

in to t h e m ed iu m , an d th e y i e ld (1 t o 2 . 5 m g p e r l i t e r )

w a s a d e q u a t e f o r a n u m b e r o f b i o l o g i c a l s tu d i e s . P e p t i de

m a p p i n g ( F i g . 3 ) i n d i c a t e d t h a t t h e r e c o m b i n a n t p r o t e i n

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

K o iv u (1 9 8 7 ) an d L ees an d B u l l e id (1 9 9 4 ) . A l so , t h e

p r o t e i n h a d a c o n f o r m a t i o n s i m i l a r t o t h a t o f t h e n a t i v e

C - p r o p e p t i d e o f t y p e I p r o c o l l a g e n a s a s s a y e d b y C D .

H o w e v e r , t h e t y p e I I I C - p r o p e p t i d e w a s s l i g h tl y m o r e

s t a b l e t o h e a t d e n a t u r a t i o n , p e r h a p s b e c a u s e i t i s a h o -

m o t r i m e r a n d t h e t y p e I C - p r o p e p t i d e i s a h e t e r o t r i m e r .

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

t i de o f t y p e I p r o c o l l a g e n w e r e f o u n d i n p a t i e n t s w i t h o s -

t e o g e n e s is i m p e r f e c t a ( se e K u i v a n i e m i e t a l . , 1 9 9 7 ) . T h e

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

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

p r o t e i n h a v e n o t b e e n d e f in e d . T h e d e v e l o p m e n t o f re -

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

t h e r m a l s t a b i l i t y o f t h e C - p r o p e p t i d e s w i l l f a c i l i t a t e t h e

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

d o m a i n s w i t h i n t h e p r o t e i n .

O f s p e c i a l i n te r e s t i s t h a t t w o p r e v i o u s r e p o r t s ( T o m i t a

e t a l . , 1 9 9 5 ; L am b er g e t a l . , 1 9 9 6 ) d esc r ib ed sy n th e s i s o f

f u l l- l e n g th t y p e I I I p r o c o l l a g e n i n t h e b a c u l o v i r u s s y s -

t e m . T h e r e c o m b i n a n t p r o t e i n f o r m e d t h e c o r r e c t d i s u l -

f i de b o n d s , b u t i t w a s r e t a i n e d i n t r a c e l l u la r l y a n d n o s i g -

n i f i c a n t a m o u n t s w e r e s e c r e t e d . T h e r e f o r e , i t w a s d i f f i -

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

d i g e s t i o n s t h a t d e g r a d e t h e N - a n d C - p r o p e p t i d e s . R e -

c o v e r y o f t h e t y p e Il l C - p r o p e p t i d e f r o m t h e m e d i u m

u n d e r t h e c o n d i t i o n s d e s c r i b e d h e r e i s p r o b a b l y e x -

p l a i n e d b y t h e s m a l l e r s i z e o f t h e p r o t e i n a n d p e r h a p s a

l e ss e r t e n d e n c y t o a g g r e g a t e .

eferences

Ala-K okko, L, Kontusa ari, S. , Baldwin, C. , Kuivaniemi, H . and

Prockop , D.J.: Structure of cDN A clones coding for the entire

prepro0tl(III) chain of hum an type III procollagen. Differ-

ences in protein structure fr om type I procollagen and conser-

vation of codon preferences.

Biochem. J. 260:

508-517 ,

1989.

Aycock, R.S., Raghow, R., Stricklin, G.P., Seyer, J.M . a nd Kang,

A.H.: Post-tr ansc ription al inhibition of collagen and fi-

bronectin synthesis by a synthetic homolog of a portion of

the carboxy-terminal propeptide of human type I collagen. J.

Biol . Chem. 261:

14355-14360, 1986.

B_chinger, H.P., Bruckner, P., Timpl, R., Prockop, D.J. and

Engel, J.: Folding Mec hanism of the triple helix in type III

collagen and type Ill pN-collagen.

Eur . J. Bioch em. 106:

619-632 (1980)

Bernard, M .P., Chu, M .L., Myers , J.C., Ram irez, E, Eikenberry,

E.E and P rockop, D.J. : Nucleotide sequences of complem en-

tary deoxyribonucleic acids for the pro0~ l chain of hu man

type I procollagen. Statistical evaluati on of structures th at are

conserved during evolution.

Biochemis t ry

22: 5213-5223,

1983a.

Bernard, M .P., M yers, J.C., Chu , M.L., Ramirez, E, Eikenberry,

E.E and Prockop, D.J. : Structure of a cDNA for the proa2

chain of hum an type I procollagen. Com pariso n with check

cD NA for protx2(I) identifies structurally conserved features

of the protein and the gene.

Biochemistry 22:

1139-1145 ,

1983b.

Bradford, M.M .: A rapid and sensitive method for the quantita-

tion of m icrogra m quantities of protein util izing the principle

of protein-dye binding.

Anal. Biochem.

72: 248-254, 1976.

Brown, M. and Falkner, P.: A plaque assay for nuclear polyhe-

drosis viruses using a solid overlay.

J. General Virology

36:

361-364, 1977.

Chen, G .C. and Yang , J.T.: Two p oint calibra tion of circular

dichrometer with d-10-camphorsulfonic acid.

Anal. Lett. 10:

1195-1207 , 1977 .

Chen, Y.-H., Yang, J.T. and Ma rtinez, H.M .: Determ ination of

the secondary structures of proteins by circular dichroism

and optical rotary dispersion.

Biochemis t ry

1 I : 4120-4131 ,

1 9 7 2

Chipm an, S .D., Sweet, H.O., McBride, J.D.J., Davisson, M.T. ,

Marks, J.S.C., Shuldiner, A.R., Wenstrup, R.J., Rowe, D.W.

and Shapiro, J.R.: Defective proc~2(I) collagen synthesis in a

recessive mutation in mice. A model of human osteogenesis

imperfecta. Proc. Natl . Acad. S ci . 90: 1701-1705, 1993.

Choglay, A.A., Purdom, I.F. and H ulmes, D.J.: Procollagen bind-

ing to sphingomyelin.

J . B io l . Chem. 268:6107-6114,

1993.

Deak, S.B. , Nicholls, A . , Pope, F.M. and Proc kop, D.J. : The

molecular defect in a nonlethal variant of osteogenesis im per-

recta. Synthesis of procz2(I) chains w hich are not in corpo -

rated into trimers of type I procollagen. J. Biol. Chem. 258:

15192-15197, 1983.

Dion, A.S. and M yers, J.C.: C OO H-te rmin al propeptides of the

ma jor hum an procollagens. Structural, functional and genetic

comparisons .

J. Molec. Biol. 193:

127-143, 1987.

Halila, R. and Peltonen, L.: Purification of human procollagen

type III N-proteinase from placenta and preparation of anti-

serum.

Biochem . J. 239:

47-52, 1986.

Hojim a, Y., McKenzie, J. , van der Rest, M. and Prockop, D.J. :

Type I procollagen N-protein ase fro m chick embr yo tendons.

Purification of a new 500-kD a form of the enzyme and iden-

tification of the catalytically active polypeptides. J. Biol.

Chem. 264:

11336-11345, 1989.

Hojim a, Y., van der Rest, M. a nd P rockop, D .J.: Type I procol-

lagen carboxyl-terminal proteinase from chick embryo ten-

dons. Purification and characterization.

J. Biol. Chem. 260:

15996-16003, 1985.

Kata yam a, K., Seyer, J.M., Raghow, R. an d K ang, A.H.: Regu-

lation of extracellular ma trix produ ction by a chemically syn-

thesized subfragm ent of type I collagen ca rboxy peptide.

Bio-

chemis t ry 30: 7097-7104,

1991.

Kessler, E. , Takahar a, K . , Biniaminov, L. , Bruse l, M. and

Greenspan , D.S . : Bone morphogen ic p ro tein - l : The type I

procollagen C-proteinase.

Science 271:

360-362, 1996.

Koivu, J. : Identification of disulfide bonds in carboxy-ter mina l

propeptides of human type I. FEBS Let ts . 212: 229-232 ,

1987.


9/9

R e c o m b i n a n t C - P r o p e p t i d e I I I 2 0 9

Kuivan iemi , H. , T romp, G. and P rockop , D.J . : Muta t ions in

f ibr i l lar col lagens ( types I , I I , II I and XI) , f ibr i l -associated col-

lagen ( type IX), and network-forming col lagen ( type X) cause

a spectrum of diseases of bone, car t i lage, and blood vessels .

H u m . M u t .

9: 300 -315 , 1997.

Lamande, S.R., Cressler , S .D., Golub, S.B., Byers , P .H., Chan,

D., Cole , W.G., Si l lence, D.O. and Bateman, J .E: Endoplas-

mic re t iculum-mediated qual i ty control of type I col lagen

product ion by cel ls f rom osteogenesis imperfecta pat ients

with mutat ions in the proixl(I) chain carboxyl- terminal

propept ide which impair subunit assembly.

J . Bio l . Chem.

270: 8642-8649 , 1995 .

Lamberg, A., Helaakoski , T., Myllyharju , J . , Pel tonen, S., Not-

bohm, H., Pihlajaniemi, T. and Kivir ikko, K.I . : Character iza-

t ion of hum an type III col lagen expressed in a baculovirus sys-

tem. Product ion of a protein with a s table t r iple hel ix required

coexpression with the two types of recombinant prolyl 4-hy-

droxylase subunit . J . B io l. C h e m . 2 7 1 : 1 1 9 8 8 - 1 1 9 9 5 , 1996.

Lees, J.E and Bulleid, N .J.: The role of cysteine residues in the

fo ld ing and a s soc ia t ion o f the COO H- te rm ina l p ropep t ide o f

type I and III procol lagen. J . Bio l . Chem. 269: 2 4 3 5 4 - 2 4 3 6 0 ,

1994.

Li, S.-W., Sieron, A.L., Fertala, A., Hojima, Y., Arnold, W.V.

and Prockop, D.J .: The C-proteinase that processes procol la-

gens to f ibr i l lar col lagens is ident ical to the protein previously

ident i f ied as bone morphogenic protein-1.

Proc . Na t l . Acad .

Sci. USA 93: 5127-5230 , 1996 .

Nichol ls , A.C., Pope, EM. and Schloon, H.: Biochemical het-

erogenei ty of osteogenesis imperfecta . New variant .

Lance t 1 :

1193, 1979.

O'Rei l ly , D.R., Mil lder , L.R. and Lucknow, V.A.: Baculovirus

expression vectors : A laboratory manual . Vol . 1 . New York:

W. H. Freeman & Co., 1992.

Prockop, D.J . and Hulmes, D.J .S.: Assembly of col lagen f ibr i ls

d e n o v o

f rom soluble precursors: Polymerizat ion and copoly-

mer iza t ion o f p roco l lagen , pN-co l l agen , and muta ted co l la -

gens. In

E x t r a c e l l u l a r M a t r i x A s s e m b l y a n d S t r u c t u r e ,

Yurchenco, P.D., Birk, D.E., Mechan, R.P., eds . , Academic

press , New York , pp . 47 -90 , 1994 .

Prockop , D.J . and Kivir ikko, K.I. Collagens: Mole cular biol-

ogy, diseases and potent ia ls for therapy.

Ann . Rev . Biochem.

64:

403-434 , 1995 .

Saiki, R.K., Scharf , S . , Faloona, E, M ull is, K.B., Ho rn, G.T., Er-

l ich, H.A. and Arnheim, N.: Enzymatic amplif icat ion of b-

globin genomic sequences and restr ic t ion s i te analysis for di-

agnosis of sickle cell anemia.

Sc ience 230:

1350-1354 , 1985 .

Sam brook , J . , Fri tsch, E.E, Man iat is , T.: Molec ular Cloning. A

Labora to ry Manua l , 2nd ed . Co ld Spr ing Harbor Labora to ry

Press , Cold Spring Harbor , 1989.

Schofie ld , J .D., Uit to , J . and Prockop, D.J .: Formation of inter-

chain disulf ide bonds and hel ical s t ructure during biosynthe-

s is of procol lagen by embryonic tendon cel ls .

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

13: 1801-1806 , 1974 .

Tomita , M., Ohkura, N., I to , M., Kato, T., Royce, P.M. and Ki-

t a j ima , T . B iosyn thes i s o f r ecombinan t human p roa l ( I I I )

chains in a baculovirus expression system: product ion of

disulphide-bonded a nd non-disulphide-b onded species con-

taining full-length triple helices.

Biochem. J . 312:

847-853 ,

1995.

Uit to , J . and Prockop, D.J .: Incorporat ion of prol ine analogues

into col lagen polypept ides . Effects on the product ion of ex-

tracel lular procol lagen and on the s tabi l i ty of the t r iple-hel i -

cal s t ructure of the molecule .

Bioch im. Biophys . Ac ta 336 :

234-251 , 1974a .

Uit to , J . and Prockop, D.J .: Intracel lular hydroxylat ion of non-

hel ical protocol lagen to form tr iple-hel ical procol lagen and

subsequent secret ion of the molecule . Eur. J . Biochem. 43:

221-230 , 1974b .

Weston, S.A., Hulmes, D.J .S., Mould, A.P., Watson, R.B. and

Humphries , M.J .: Ident i f icat ion of integrin a21bl as cel l sur-

face receptor for the carboxy-terminal propept ide of type I

procol lagen. J . Bio l . Chem. 269: 20982-20988 , 1994 .

Wu, C.H., Donovan, C.B., and Wu, G.Y.: Evidence for pre-

t ransla t ional regulat ion of col lagen synthesis by procol lagen

propept ides .

J . Bio l. Ch em. 261 :

10482-10484 , 1986 .

Wu, C .H. , Wal ton , C .M. and Wu, G.Y. : P ropep t ide -med ia ted

regulat ion of procol lagen synthesis in IMR-90 human lung f i -

broblast cel l cul tures . Evidence for t ranscr ipt ional control . J .

Bio l . Chem.

266 : 2983-2987 , 1991 .

Darwin J . Prockop, Center for Gene Therapy, Allegheny Uni-

vers i ty of the H eal th Sciences, Phi ladelphia , PA 19102

Received June 5, 1997; accepted July 17, 1997

1997-KHA3A ZAFARULLAH-Synthesis and Conformational Properties.pdf

Documents

Transcript of 1997-KHA3A ZAFARULLAH-Synthesis and Conformational Properties.pdf