1997-WILLIAM V. ARNOLD- A cDNA Cassette system for the synthesis of recombinant procollagens.pdf

8/10/2019 1997-WILLIAM V. ARNOLD- A cDNA Cassette system for the synthesis of recombinant procollagens.pdf

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, , : : , ~ ', ,, Z , -~ D ,

A c D N A C a s s e t te S y s te m f o r t h e S y n t h e s is

o f R ecom binant Proco l lagens . Var ian ts

o f P roco l lagen I I Lack ing a D Per iod Are Secre ted

as Tr ip le He l ica l Monomers

W ILL IAM V. ARNOLD I , ALEKSANDER L . S IERON I , ANDRZE J FERTALA I ,

HANS PETER BAC HINGER i ', D IANE MEC HLINGi a nd DAR W IN O. PROCKOP I

Department of Biochemistry and Molecular B iology,Jefferson Institute of Mo lecular M edicine, Jeffer-

son Medical College of T homas Jefferson U niversity, Philadelphia, Pennsylvania and

t Shriners Hospital for C hildren and Department of Biochemistry and Molecular Biology, Oregon

He alth Sciences University, Portland, Oregon , USA.

A b s t r a c t

C u r r e n t l y t h e r e i s a l a c k o f e x p e r ime n ta l s y s t e ms f o r d e f in in g th e f u n c t i o n a l d o m a in s o f t h e

f i b r i l l a r c o l l a g e n s . H e r e w e d e s c r i b e a n e x p e r ime n ta l s t r a t e g y t h a t e mp lo y s t h e p o ly me r a s e

cha in r eac t ion (PCR) to c rea te a se r ie s of cDNA casse t te s coding for seven sepa ra te domains

o f p r o c o l la g e n I I. T h e s y s t e m w a s u s e d t o p r e p a r e n o v e l r e c o m b in a n t p r o c o l l a g e n s I f r o m

w h ic h o n e o f t h e f o u r r e p e ti t iv e D - p e r io d s o f t h e t r i p le h e l i x w a s d e l e te d . F o u r c o n s t r u c t s ,

e a c h l a c k in g a d i f f e r e n t D - p e r io d , w e r e e x p r e s s e d i n s t a b ly t r a n s f e c t e d ma mma l i a n c e l l s

( H T - 1 0 8 0 ) . T r u n c a t e d p r o c o l l a g e n s o f t h e p r e d i c t e d s iz e w e re r e c o v e r e d f r o m th e m e d iu m.

Al l were t r ip le -he l ica l a s a ssayed by c i r cu la r d ichro ism. There fore , de le t ion of a comple te

D - p e r io d c o n t a in in g 2 3 4 a min o a c id s d o e s n o t d e s t a b i l i z e t h e t r i p l e h e l i x o f h o mo t r ime r i c

c o l l a g e n II as mu c h a s s o me n a tu r a l l y o c c u r r i n g mu ta t i o n s i n t h e h e t e r o t rime r i c mo n o m e r o f

co l lagen I tha t de le te shor te r sequences or tha t conver t ob l iga te g lyc ine r e s idues to r e s idues

wi th bulk ie r s ide cha ins . Moreover , the r e su l t s sugges t tha t the s t r a tegy deve loped he re can

b e u s e d t o ma p i n d e t a il t h e b in d in g s i te s o n f i b r i ll a r co l l a g en s f o r o th e r c o mp o n e n t s o f t h e

ext race l lu la r ma t r ix and for the b inding , spread ing an d s igna l ing of ce ll s.

K e y w o r d s : c D N A c a s s et t es , r e c o mb in a n t t y p e I I p r o c o l l a g e n

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

C o l l a g e n s a r e a m a jo r f a mi ly o f e x t r ac e l l u l a r p r o t ein s

th a t h a v e a v a r i e ty o f f u n c t i o n s a n d t h a t a r e c h a r a c t e r -

1 Current address: Cen ter for Gen e Therapy, Allegheny Univer-

sity of the Health Sciences, 245 No rth 15 Street, M .S. 421,

Philadelphia, PA 19102

M atr ix B io logy Vol. 16 /1997, pp . 105-1 16

1997 by Gustav Fischer Verlag

ized by having a t leas t one reg ion in which three

polypept ide cha ins a re fo lded in to a cha rac te r i s t ic

t r ip le -he l ica l con form at io n (P iez, 1984; van de r Res t and

G a r r o n e , 1 9 9 1 ; P r o c k o p a n d H u lme s , 1 9 9 4 ; P r o c k o p

a n d K iv i r i k k o , 1 9 9 5 ) . F o ld in g o f t h e p r o t e in i n to t h e

t r ip le -he l ica l conformat ion i s d r iven by repe t i t ive se -

q u e n c e s o f - G ly - X x x - Y y y - t ri p e p t i d e u n i t s i n w h ic h

-Xxx- i s f r equent ly pro l ine and -Yyy- i s f r equent ly hy-


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1 0 6 W . V . A r n o l d e t a l .

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

f i b r i l - f o r m i n g c o l l a g e n s k n o w n a s t y p e s I , I I a n d I I I .

E a c h o f t h es e h a s a m a j o r t r i p le - h e li c a l d o m a i n o f a b o u t

1 , 0 0 0 r e s i d u e s , a n d e a c h i s f i r s t s y n t h e s i z e d a s a s o l u b l e

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

C - p r o p e p t i d e s ( F i g . 1 ) . A f t e r t h e p r o p e p t i d e s a r e c l e a v e d

b y s p e c if ic N - a n d C - p r o t e i n a s e s , t h e c o l l ag e n m o l e c u l e s

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

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

a m i n o a c i d s in t h e m o n o m e r s o f c o l la g e n I a n d I I in d i-

c a t e d t h a t t h e r e p e a t i n g t r i p e p t i d e u n i t s o f 1 , 0 1 4 a m i n o

a c i d s a r e d i v i d e d i n t o 4 . 4 r e p e a t s o r 4 . 4 D - p e r i o d s o f

a b o u t 2 3 4 a m i n o a c i d s e ac h ( H u l m e s e t a l ., 1 9 7 3 ; C h a p -

m a n , 1 9 8 4 ) . I n f i b r i l s , t h e m o n o m e r s a r e a r r a n g e d i n .

a h e a d -t o - t a il o r i e n t a t i o n w i t h a g a p o f a b o u t 0 . 6 D -

p e r i o d s a n d , t h e r e f o r e , a r e p e a t o f 5 D - p e r i o d s .

T h e m o n o m e r s o f th e f i b r il l a r c o l la g e n s w e r e i n i ti a l ly

r e g a r d e d a s ri g id r o d s . S u b s e q u e n t o b s e r v a t i o n s , h o w -

e v er , p r o v e d t h a t s h o r t s eg m e n t s o r c o o p e r a t i v e

b l o c k s ( P r iv i lo v , 1 9 8 2 ) o f t h e t ri p l e h e l ix b e g i n t o u n -

d e r g o m i c r o - u n f o l d i n g a t a b o u t 3 0 t o 3 7 C a n d w e l l b e -

f o r e th e m o l e c u l e f u ll y u n f o ld s a t a b o u t 4 1 C . E v i d e n c e

f o r t h e m i c r o - u n f o l d i n g m o d e l i n c l u d es t h e e f f e ct s o f

p a r t i a l l y d e n a t u r i n g a n d t h e n r e n a t u r i n g t h e p r o t e i n

( K i i h n e t a l , 1 9 6 6 ; R h y ~ i n e n e t a l . , 1 9 8 3 ) , c o m p a r i s o n s

o f t h e h e l i x - fo r m i n g p r o p e r t i e s o f s y n t h e ti c p e p t i d e s

w i t h r e p e t it i v e - G l y - X x x - Y y y - s e q u e n c e s ( S a k a k i b a r a e t

a l . , 1 9 7 3 ; P r o c k o p e t a l . , 1 9 7 6 ; E n g e l e t al . , 1 9 7 7 ; I n -

o u y e e t a l. , 1 9 8 2 ; D 61 z a n d H e i d e m a n n , 1 9 8 6 ; R o t h a n d

H e i d e m a n n , 1 9 8 6 ; G e r m a n n a n d H e i d e m a n n , 1 9 8 8 ) ,

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

t i o n b y m i c r o c a l o r i m e t r y ( P r i v a lo v , 1 9 8 2 ) a n d t h e e f f e ct s

o f te m p e r a t u r e o n t h e k i n e t i cs o f f ib r il f o r m a t i o n

( K a d l e r e t a l . , 1 9 8 8 ) . T h e r e s u l t s d e m o n s t r a t e d t h a t s e -

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

l i ne , a n d e s p e c i a l l y s e q u e n c e s i n w h i c h t h e Y y y p o s i t i o n s

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

t r i p l e - h e l i c a l r e g i o n s . R e g i o n s l a c k i n g t h e t w o i m i n o

S i g n a l P e p t i d e

C t e l o p e p t i d e

N P r o p e p t i d e T r i p le

N t e l o p e p t i d e H e l i x C P r o p e p t i d e

N P r o t e i n a s e C P r o t e in a s e

C l e a v a g e S i t e C l e a v a g e S i t e

N t D 1 D 2 D 3 D 4 D 0 . 4 C t

1 3 7 a a 2 3 4 a a 2 3 4 a a 2 3 4 a a 2 3 4 a a 78 a a 2 7 3 a a

Figure 1. Schematic drawing o f Procollagen II. Th e subdivisions of the protein indicated belo w the molecule we re used in the de-

sign of the procollagen II DN A cassette system. Sym bols: aa, am ino acids.


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

m u c h l o w e r t e m p e r a t u r e s . H o w e v e r , i t i s u n l i k e l y t h a t

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

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

t r i p l e h e l i x w i t h a b u l k i e r a m i n o a c i d c a n l o w e r t h e

m e l t i n g t e m p e r a t u r e b y a s m u c h a s 2 0 C ( W e s t e r h a u s e n

e t a l . , 1 9 9 0 ; K u i v a n i e m i e t a l . , 1 9 9 6 ) . A l s o , t h e l o c a t i o n s

a n d s i z e s o f t h e c o o p e r a t i v e b l o c k s h a v e n o t b e e n d e -

f ined .

H e r e w e r e p o r t a n e x p e r i m e n t a l s t r a t e g y t h a t g r e a t l y

s i m p l if i es m a p p i n g t h e f u n c t i o n a l d o m a i n s o f f i b ri l la r

c o l l a g e n s . W e h a v e c r e a t e d a s e r i e s o f c D N A c a s s e t t e s

t h a t c o d e f o r t h e i n d i v i d u a l D - p e r i o d s a n d t h e N - a n d

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

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

o r d e r i n t o D N A c o n s t r u c t s t h a t c o d e f o r n o v e l v a r i a n t s

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

A c D N A C a s s e t te S y s t em 1 0 7

m u t a t e d b e f o r e a s s e m b l y . W e h a v e a s s e m b l e d f o u r c o n -

s t r u c ts t h a t e a c h l a c k a s p e c if ic D - p e r i o d a n d h a v e

s h o w n t h a t w e c a n e x p r e s s t h e m a s t r u n c a t e d v a r i a n t s o f

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

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

s u it s i n d ic a t e t h a t d e l e t i o n o f a c o m p l e t e D - p e r i o d o f

2 3 4 a m i n o a c i d s h a s s u r p r i s i n g l y l i tt l e e f f e c t o n t h e t h e r -

m a l s t a b i l i t y o f c o l l a g e n I I .

Materials and Methods

Design o f the procollagen II D N cassette system

c D N A c a s s e t t e s w e r e s y n t h e s i z e d a s i n d i c a t e d i n F i g -

u r e s 2 a n d 3 , u s i n g P C R w i t h a t e m p l a t e o f a f u l l- l en g t h

c D N A f o r p r o c o l l a g e n I I ( B a l d w in e t a l. , 1 9 8 9 ; c o u r t e s y

5 6 6 6 b b b 3

c c c c c

P C R

S 3

L I G T E

c a s s e t t e

B i n s e r t

Figure 2. Cre ation o f a DN A cassette. PCR p rime rs containing engineered blunt-cutting restr iction sites B and C w ere used to

PCR-subclone a region of a cDNA template. The restr iction site A was a part of the multiple cloning region of the plasmid vector.

The A nucleotide show n at the 3 -ends of the PCR pro duct w ere added by Taq polym erase and prov ided nucleot ide overhangs used

in the TA cloning system (Invitrogen).


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1 0 8 W . V . A r n o l d e t a l.

B . R e a i o n 1 B .

5 - 5

C A C

A

c l e a v e a t s i t e s / / c l e a v e a t s i t e s

A a n d C A a n d B

3 C

B

l e a i o n 2

A

Figure 3 . D NA cons t ruc t assembly us ing the DN A casset te sys tem. T he assemb ly of a cons t ru c t conta ining tw o casset te inser ts i s

dem onst ra ted . The A , B and C res t r i c tion s i tes a re the same in the three p lasmids shown . The nucleot ide sequences o f some s it es

are capi ta l ized so tha t th ey m ay be di f ferent ia ted f ro m the sam e s it es in d i f ferent p lasmids .

o f S .- W . L i ). T h e P C R p r i m e r s w e r e e n g i n e e r e d t o i n t r o -

d u c e b l u n t - c u t t i n g r e s t r i c t i o n s i te s B a n d C F i g . 2 ) a t t h e

e n d s o f t h e a m p l i f i e d r e g i o n . T h e s e r e s t r i c t i o n s i te s F i g .

2 a n d 3 ) w e r e d e s i g n e d t o m e e t th e f o l l o w i n g c r i te r i a : a )

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

e n c o d e d b y t h e c D N A ; b ) r e s t r ic t i o n s i te B d i d n o t

o c c u r i n th e w i l d - t y p e c D N A t e m p l a t e u s e d t o c r e a te t h e

c a s s e t t e s o r i n t h e p l a s m i d v e c t o r s ; c ) r e s t r i c t i o n s i t e C

i n a g i v e n D N A c a s s e t te w a s u n i q u e f o r th e c a s s e tt e i n

w h i c h i t w a s u s e d s i te A to s i te C i n P l a s m i d I o f F i g . 3 )

b u t c o u l d o c c u r a n y w h e r e i n t h e D N A s e q u e n c e o f o t h e r

D N A c a s s e tt e s ; a n d d ) r e s t r i c ti o n s i te A w a s a st i c k y -


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

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

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

D i f f e r e n t D N A c a s s e t t e s w e r e a s s e m b l e d t o c r e a t e

l a r g e r D N A c o n s t r u c t s a s i n d i c a t e d i n F i g u r e 3 . P la s m i d

I w a s d e f i n e d a s a p - c a s s e t t e b e c a u s e i t p r o v i d e d t h e

D N A c a s s e t te t h a t w a s i n s e r t e d a t t h e 5 ' e n d o f th e c a s -

s e t t e in P l a s m i d I I . P l a s m i d I I w a s d e f i n e d a s a n r - c a s -

s e t t e b e c a u s e i t r e c e i v e d a D N A i n s e r t f r o m a p - c a s s e t t e .

S y n t h e s i s o f D N A c a s s et te s

S e v e n D N A c a s s e t t e s w e r e c r e a t e d t o c o d e f o r s e v e n

s e p a r a t e r e g i o n s o f t h e p r o c x l (I I ) c h a in o f t y p e I I p r o c o l -

l a g e n (F ig . 1). B e c au s e t h e 5 e n d o f t h e C O L 2 A 1 c D N A

w a s d i f f i c u l t t o i s o l a t e ( B a l d w i n e t a l . , 1 9 8 9 ) , n u -

c l e o t i d e s 2 3 t o 1 2 6 ( n u c l e o t i d e 2 3 i s t h e f i r s t o f th e s t a r t

c o d o n ) i n t h e c o n s tr u c t w e r e f r o m e x o n 1 o f h u m a n

C O L I A 1 c D N A ( T r o m p et a l. , 1 9 8 8 ). T h e r e m a i n d e r o f

t h e c D N A , n u c l e o t i d e s 1 2 7 t o 4 7 3 7 , w e r e d e r i v e d f r o m

e x o n s 3 t o 5 2 o f h u m a n C O L 2 A 1 g e n e ( B a ld w i n e t a l. ,

1 9 8 9 ; A l a - K o k k o a n d P r o c k o p , 1 9 9 0 ; F e r t a l a e t a l .,

1 9 9 4 ) . T h e r e f o r e , t h e c o n s t r u c t i n c l u d e d t h e s i g n a l p e p -

t i d e a n d s i g n a l p e p t i d e c l e a v a g e s i t e f r o m t h e C O L 1 A 1

g e n e . H o w e v e r , i t d i d n o t i n c lu d e e i t h e r e x o n 2 f r o m t h e

A c D N A C a s s e t t e S y s t em 1 0 9

C O L I A 1 o r e x o n 2 A o r 2 B f r o m t h e C O L 2 A 1 g e n e , t h e

e x o n s t h a t a r e a l t e r n a t i v e l y s p l i c e d i n t h e C O L 2 A 1 g e n e

( P r o c k o p a n d K i v i r i k k o , 1 9 9 5 ) . T h e P C R s w e r e c a r r i e d

o u t w i t h a c o m m e r c i a l k it ( G e n e A m p P C R R e a g e n t K i t;

P e r k i n E l m e r ) w i t h 0 . 2 n g /l al c D N A f o r p r o ~ l ( I I ) c h a i n s

a s t e m p l a t e , p r i m e r s ( E x p r e s s G e n e t i c s , P r i n c e t o n , N J ) i n

a c o n c e n t r a t i o n o f 0 . 4 p m o l / l a l ( T a b l e I ) , a n d o n e - n i n t h

v o l u m e o f 5 m M M g C I 2. T h e c o n d i t i o n s w e r e : 9 4 . 5 C

f o r 1 m i n f o l l o w e d b y c y c l e s o f 9 4 C f o r 3 sec; 5 5 C

f o r 3 0 s e c a n d 7 2 C f o r 6 0 s e c . A f t e r 3 c y c l e s , t h e s a m -

p l e s w e r e a n n e a l e d a t 7 2 C f o r 4 2 0 s ec a n d t h e n s t o r e d

a t 4 C . T h e P C R p r o d u c t s w e r e l i g a t e d i n t o a T A

c l o n i n g v e c t o r ( P C R I I ; I n v i t r o g e n ) 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 in s t r u c t i o n s ( Fi g. 4 a ) . T h e s t r u c t u r e o f e a c h

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

s e q u e n c i n g ( N u c l e i c A c i d F a c i l i t y , K i m m e l C a n c e r I n s t i -

t u t e , T h o m a s J e f f e r s o n U n i v e r s i t y , P h i l a d e l p h i a , P A ) .

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

t h e n t r a n s f e r r e d t o t h e v e c t o r p c D N A I I ( I n v i tr o g e n ) b y

d i g e s t i o n w i t h H i n d l I I a n d S p h I ( F i g . 4 b ) , f o l l o w e d b y

l i g a t i o n w i t h T 4 D N A l i g a s e ( L if e T e c h n o l o g i e s , I n c .) .

T o e n s u r e e x p r e s s i o n , a 2 - k b P p u M I / P u v l I f r a g m e n t o f

t h e C O L 2 A 1 g e n e e n c o d i n g t h e 3 ' u n t r a n s l a t e d r e g i o n

( U T R ) ( B a l dw i n e t a l ., 1 9 8 9 ; c o u r t e s y o f L. A l a - K o k k o )

w a s i n s e r t e d i n t o t h e c o m p a t i b l e P p u M I / E c o R V s i t e a t

Table I . PCR Pr imers used for subcloning the pro~ l( I I ) cD NA in the creat ion of the proct l( I I ) DN A casset te sys tem.

Prime r Prime r Sequence b Engineered

N a m e a Restriction Sitec

N ,+ G T C T A C A T G T C T A G G G T C T A G A C A T G ' Y I' C A G N O N E

N ~ - C A G / C T G C A T T A C T C C C A A C T G G G C G C C A C C A P v u l I (C)

D I + G C C C / G G G C C A A T G G G C C C C A T G G G A C C T C G Sr[I (B)

D 1 - G A G / C G G G A A G C C A G G A G C A C C A G C A A T G C C B srB I (C)

D 2 + G C C C / G G G C C A C G G G G T C C T C C T G G C C C T C A Srf l (B)

D 2 - C A G / C G G A A G T C C C T G G A A C C C A G A T G G C C C B srB I (C)

D 3 + G C C C / G G G G C T C C T G G T C C C C C A G G T G A A G G T Srf l (B)

D 3 - C G / C G C A G C T C C A G G G A A T C C A G T G G C T C C C G B s tU I (C)

D 4 + G C C C / G G G C G T G T T G G A C C C C C A G G C T C C A A T G

Srf l

(B)

D 4 - C G / C G T G A A G C C A C G G T G T C C C T I ' C A G G C C T C T B s tU I (C)

D0.4 G C C C / G G G C T G C A G G G T C T G C C C G G C C C T C C T G G T C S rfI (B )

D0.4-

G A G / C G G G G G A C C T G G A G G A C C A G G G G G C C C A G G A T B srB I (C)

C t + G C C C / G G G C C T G G C A T C G A C A T G T C C G C C T

Srf I

(B)

C t - T C T G G C C T G G G C T G G G G G C A G T C A C T C A G N O N E

a The pr imer name denotes the region of the procxl(I I) cDN A that this pr imer was used to subclone by PCR. A + or - in the

pr imer na me denotes whe ther the pr imer pr imes the p olyme r izat ion of the sense or the ant isense s trand, respect ively.

b All prim ers are written from the 5 ' to 3' end. Boldface nucleotides indicate wh ere changes were made fro m the original pro0 tl(II)

cDN A. These changes we re engineered to introduce the indicated restriction sites, which are underlined. Th e slash in each prim er

sequence indicates wh ere the corresp ond ing restr iction enzy me cleaves the engineered restr iction site.

' The (B) or (C) following the nam e of the engineered restr iction site indicates whe ther tha t site is used as a B or a C restr iction site

in the cor responding DN A cassette .

The ad dit ional change (C to T) introduced h ere was done to des troy a nat ive B s tU I site which occurs in this position in the origi-

nal pro00 (I I ) cDN A. W ithout this change the B s tU I site engineered at the 3' end of the D 4 c a s s e t t e wou ld no t be unique for this

coding region. Note that this C to T change does not alter the codon at this position.


6/12

110 W.V. Arno ld e t a l.

( ' )

j I IL

H in d l l /~ ~ S p h l

S p e l s r I

I k a n ~ [ a m p I

p CR I I 3 .9 kb )

b )

Sphl

/ /

I I I m p I

/ I

indlll

p cDN A I I 3 .0 kb )

c )

E c o R V P v u l

I

n o

H a m p

indlll

p c D N A 3 5 . 4 k b )

Figure 4. Plasmids used in the creation of the procollagen II

DNA cassette syste m. The plasmid vectors we re used in (a)

PCR-subcloning, (b) ass em bly of D NA cassettes into con-

structs, and (c) construct for mam malian cell transfection.

t h e 3 ' e n d o f t h e D N A c a s s e t t e e n c o d in g t h e p r o ( x l ( I I )

C - p r o p e p t i d e . T h i s 3 ' U T R r e g io n w a s s u b s e q u e n t l y

c loned a t the 3 ' end of the C t casse t te .

eD NA constructs

F iv e D N A c o n s t r u c t s ( T a b l e I I ) w e r e a s s e mb le d f r o m

the casse t te s in the o rde r in d ica ted in Table I l l , us ing the

bac te r ia l s t r a in DH5(x (Li fe Technologies , Inc . ) a s hos t .

I n e a c h a s s e mb ly s t e p , t h e f i d e li t y o f t h e D N A s e q u e n c e

a t t h e j u n c t i o n s w a s v e r i f i ed b y D N A s e q u e n c in g . F u n c -

t i o n a l c o n s t r u c t s w e r e r e mo v e d i n t a c t b y d ig e s t io n w i th

HindIII

a n d

Bs r BI

a n d c lo n e d i n to t h e c o r r e s p o n d in g

HindIII

a n d

E c o R V

s i t e s o f t h e ma mma l i a n e x p r e s s io n

v e c to r p c D N A 3 ( I n vi t ro g e n ) c o n t a in in g t h e C M V p r o -

m ote r a nd a neo m yc in re s i s tance (F ig. 4c) .

Cell transfections

H T - 1 0 8 0 c e l l s ( A me r i c a n T y p e C u l tu r e C o l l e c t i o n

C C L 1 2 1 ) w e r e c u l t u r e d i n D M E M s u p p l e m e n t e d w i t h

10 (v/v) f e ta l ca l f se rum . Th e ce l ls were t r ansfec ted

( F e r ta l a et a l. , 1 9 9 4 ) w i th o n e o f t h e D N A c o n s t r u c t s b y

c a l c iu m p h o s p h a t e p r e c ip i t a t i o n u s in g a c o mme r c i a l k i t

( P r o f e c t i o n Ma mma l i a n T r a n s f e c t i o n S y s t e m K i t ;

P r o me g a ) , a c c o r d in g t o t h e ma n u f a c tu r e r ' s i n s t r u c t i o n s .

Br ie fly , ce ll s were sp l i t 18 h pr io r to t r ansfec t ion , g ro wn

to a dens i ty of approxima te ly 106 ce l l s in a 10 mm ce l l

c u l t u r e d i s h , a n d p r o v id e d w i th f r e s h c u l t u r e me d iu m

3 h p r i o r t o t r a n s f e c t i o n . A p p r o x im a te ly 1 2 l ag o f D N A

l in e a r i z e d w i th PvuI w a s p r e c ip i t a t e d w i th c a l c iu m

p h o s p h a t e a n d i n c u b a t e d w i th t h e c e l l s . A f t e r a b o u t

1 7 h , f r e s h c u l t u re m e d iu m w a s a p p l i e d . T h e c e l ls w e r e

sp l i t 1 to 10 , and 24 h la te r cu l tured in the cu l ture

me d iu m c o n t a in in g G 4 1 8 ( L if e T e c h n o lo g i e s , I n c. ) a t a

c o n c e n t r a t i o n o f 0. 4 m g / L . T h e m e d i u m w a s e x c h a n g e d

w i th f r e s h s e l e c t i o n me d iu m e v e r y 4 8 h o v e r a 1 2 - d a y

per iod .

Screening o f transfected clones

I so la ted G418- res is tan t ce l l Colonies were expanded in

12-we l l p la te s . Upo n rea ching con f luence , cel ls were cu l -

t u r e d f o r 2 4 h w i th 1 ml o f s e r u m- f r e e D ME M s u p p l e -

me n te d w i th 4 1 i ag /ml L -a s c o r b ic a c id p h o s p h a t e ma g n e -

s iu m s a l t n - h y d r a t e ( W A K O P u r e C h e mic a l I n d u s t r i e s ,

L td) and 0 .5 laC i/ml of un i form ly 14C- labeled am ino ac id

mix tu r e ( N E N D u P o n t ) . Me d ia w e r e c o l l e c t e d a n d p r o -

t e in s p r e c ip it a t e d w i th 8 0 0 0 M W p o ly e th y l e n e g ly c o l

(S igma) a t a f ina l con cent ra t ion of 5 . P rec ip i ta ted pro-

te ins were d isso lved in s torage bu f fe r (0 .4 M NaCI ,

2 5 mM E D T A , 0 . 0 4 N a N 3 i n 0 .1 M T r is H C I , p H 7 .4 )

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 r e d u c in g c o n d i t i o n s ,

f o l l o w e d b y e l e c t r o b lo t t i n g a n d W e s t e r n a n a ly s i s u s in g

g u in e a p ig a n t i - h u m a n a n t i b o d i e s s p e c if ic t o t h e C - t elo -

pept ide r eg io n of procol lagen I I (Ala -Kok ko e t al . , 1991;

k in d ly p r o v id e d b y C . Me r r y ma n , D e p t . B io c h e m. a n d

Molec . B io l . , Thomas Je f fe r son Unive r s i ty , Phi lade lphia ,

P A ) a n d s e c o n d a r y a n t i b o d i e s a n t i - g u in e a p ig Ig G c o n ju -

ga ted wi th a lka l ine phospha tase (S igma) .

Recom binant procol lagens product ion

S e le c t e d c lo n e s w e r e g r o w n to c o n f lu e n c e i n D ME M

s u p p le m e n te d w i th t 0 ( v/v) f e t a l c a l f s e r u m in t e n

1 7 5 - c m 2 c u l t u r e f l as k s . T h e c lo n e s w e r e e x p a n d e d i n

fou r in te rcon nec t ing t i ssue -cu l ture f la sks (Ce ll Fac tor ie s ,

N u n c ) t h a t w e r e e q u iv a l e n t t o a p p r o x ima te ly o n e h u n -

d r e d t h i r t y -s e v e n 1 7 5 - c m 2 f l a sk s o f c u l t u r e d c e ll s . W h e n

th e c u l t u r e r e a c h e d a p p r o x ima te ly 8 0 c o n f lu e n c e , t h e

me d iu m w a s r e mo v e d a n d t h e c e l l l a y e r w a s w a s h e d

br ie f ly wi t h PBS. The ce ll s were then incu ba ted w i th la -

be l ing se rum-f ree medium conta in ing 0 .17 laC i /ml of a

u n i f o r m ly 1 4C -l ab el ed a m in o a c id mix tu r e a n d 4 1 p g /ml

o f L - a s c o rb i c a c id p h o s p h a t e ma g n e s iu m s a l t n - h y d r a t e .

A f t e r 2 4 h , t h e me d iu m w a s c o l le c t e d a n d r e p l a ce d w i th

f r es h l a b e li n g me d iu m . A f t e r a n o th e r 2 4 h , th i s me d iu m

w a s r e p l a c e d w i th t h e me d iu m th a t d id n o t c o n t a in r a -

d ioac t iv i ty . Fol lowing co l lec t ion of the th i rd 24-h


7/12

m e d i u m , t h e c e ll l a y e r w a s w a sh e d t w i c e b r i e f l y w i t h

P B S c o n t a i n i n g 1 m M E D T A . T h e c e l ls w e r e t h e n t r e a t e d

w i t h t h e s a m e c y c l e o f t h r e e c o n se c u t i v e 2 4 - h i n c u b a -

t i o n s o f m e d i u m .

Purification of recomb inant procollagens

T h e m e t h o d o f F e r ta l a e t a l. ( 1 9 9 4 ) w a s u s e d w i t h

m i n o r m o d i f i c a t i o n s . F o r e a c h c e l l l i n e , a p p r o x i m a t e l y

4 L o f m e d i a h a r v e s t e d f r o m e a c h 2 4 - h p e r i o d w a s f i l -

t e r e d t h r o u g h a 1 . 6 l a i n g l a s s - f i b e r f i lt e r ( M i l l ip o r e ) a n d

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

c a t e d c o n c e n t r a t i o n s : 0 . 1 M T r i s H C 1 b u f f e r , 0 . 4 M

N a C 1, 2 5 m M E D T A , 1 0 m M N E M , 1 m M P AB , a n d

0 . 0 4 % N a N 3 a d j u s t e d t o p H 7 . 4 . H i g h m o l e c u l a r m a ss

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

m a t e l y 1 0 - f o l d a t 4 C b y t h e u se o f c a r t r i d g e s w i t h

1 0 0 - k D a m o l e c u l a r m a ss c u t - o f f ( P r e p / S c a l e - T F F f i l t e r ;

M i l l i p o r e ) . P r o t e i n s i n t h e c o n c e n t r a t e d m e d i u m w e r e

p r e c i p i t a t e d o v e r n i g h t a t 4 C w i t h 2 0 0 m g / m l a m m o -

n i u m su l f a t e a n d c e n t r i f u g a t i o n a t 1 5 , 0 0 0 g f o r 1 h a t

4 C . P e l l e ts f r o m e a c h 2 4 - h c o l l e c t i o n w e r e p o o l e d , so l -

u b i l i z e d i n s t o r a g e b u f f e r o v e r n i g h t a t 4 C , a n d d i a M

l y z e d t w i c e a g a i n s t 2 0 0 v o l u m e s o f D E A E - c e l l u l o se c o l -

u m n I b u f f e r ( 2 M u r e a , 0 .2 M N a C I , 5 m M E D T A , a n d

0 . 0 4 % N a N 3 i n 0 . 1 M T r i s H C I , p H 7 . 4 ). T h e s a m p l e

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

4 C o n a D E A E - c e l l u l o se c o l u m n ( 2 . 6 c m 1 5 c m )

e q u i l i b r a t e d a n d e l u t e d w i t h t h e D E A E - c e l l u l o se c o l -

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

a n d d i a l y ze d a t 4 C a g a i n s t 2 0 0 v o l u m e s o f D E A E - c e l -

l u lo s e II c o l u m n b u f f e r ( 2 M u r e a , 2 m M E D T A , a n d

0 . 0 4 % N a N 3 i n T r is H C 1 b uf f e r, p H 7 . 8 ). T h e s a m p l e

w a s c h r o m a t o g r a p h e d a t 4 C o n a s e c o n d D E A E - c e l l u -

l o s e c o l u m n ( 2 . 6 c m 1 5 c m ) e q u i l i b r a t e d a n d e l u t e d

w i t h t h e D E A E - c e l l u l o se c o l u m n I I b u f f e r . T h e

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

a p p l i e d i n t h e s a m e b u f f e r t o a c o l u m n ( 1 . 6 c m 5 c m )

o f Q - S e p h a r o s e ( P h a r m a c i a ) a t 4 C . T h e c o l u m n w a s

w a s h e d w i t h D E A E - c e ll u l o se c o l u m n b u f f e r I I, a n d r e -

c o m b i n a n t p r o c o l l a g e n II w a s e l u t e d w i t h a 0 . 4 M N a C I

i n th e s a m e b u f f e r . T h e e l u t e d p r o t e i n w a s d i a l y z e d a t

4 C a g a i n s t 2 0 0 v o l u m e s o f s to r a g e b u f f e r c o n t a in i n g

5 m M E D T A a n d s t o r e d a t - 8 0 C . F o r f u r t h e r a na l y si s

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

o n a m e m b r a n e f i l t e r ( Y M - 1 0 0 ; A m i c o n ) , a n d t h e

b u f f e r w a s e x c h a n g e d t o E D T A - f r e e s to r a g e b u f f e r. T h e

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

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

W i s t a r P r o t e i n M i c r o c h e m i s t r y C o r e F a c i l i t y , P h i l a d e l -

p h i a , P A .

A c D N A C a sse t te S y s t e m 1 1 1

CD spectroscopy o f recombinant procollagens

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

w i t h a J A S C O J - 5 0 0 A s p e c t r o p o l a r i m e t e r u s i n g w a t e r -

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

0 . 0 5 c m ( H e l lm a ) . T h e t e m p e r a t u r e o f th e s a m p l e w a s

m o n i t o r e d b y a t h e r m i s t o r a n d a d ig i ta l t h e r m o m e t e r

( O m e g a E n g i n e e r i n g , In c . ). T h e t e m p e r a t u r e o f t h e c ir -

c u l a t i n g w a t e r b a t h ( L a u d a R C S 2 0 D ) w a s c o n t r o l l e d b y

a t em p e r a t u r e p r o g r a m m e r ( L a u d a P M 3 5 0 ) . T h e C D

s p e c t r u m o f th e s a m p l e w a s s c a n n e d f r o m 1 8 0 n m t o

2 6 0 n m .

esutts

General features o f the cassette system

I n d e s i g n i n g c a s se t t e s f o r t h e p r o ~ l ( I I ) c h a i n , t h e

8rfl

( 5 ' - G C C C / G G G C - 3 ' ) s i t e w a s u se d a s t h e B s i t e ( F i g . 2 )

for a l l t he casse t t e s because Srlq s i t e s a re ra re in p l a smid

v e c t o r s , d o n o t o c c u r i n p r o t x l ( I I ) c D N A , a n d c l e a v a g e

o f t h e s it e l e a v es a c o m p l e t e c o d o n f o r g l y c i n e ( G G N ) a t

t h e 5 e n d o f t h e c a s se tt e . A c c o r d i n g l y , a

Srfl

s i t e can be

u se d t o d e f i n e t h e 5 ' e n d o f a D - p e r i o d o r b u i l d a n y c a s -

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

a n y w h e r e w i t h i n a D - p e r i o d o f th e p r o ~ l ( I I ) c h a i n . T o

a c c o m m o d a t e t h e v a r i ab i l it y i n t h e - Y y y - p o s i ti o n s t h a t

e n d e a c h D - p e r i o d , t h e r e s t r i c ti o n s e q u e n c e s i n t r o d u c e d

i n t o t h e C s i t e w e r e Bs r BI ( G A G / C G G ) f o r p r o l i n e

( C C N i n t h e 3 ' t o 5 ' o r i e n t a t i o n ) , BstUI ( C G / C G ) f o r

a l a n i n e ( G C N ) , a n d

Eco 7III

( A G C / G C T ) f o r s e r i n e

( A G N ) .

T h e N t a n d C t r e g io n s c o n t a i n i n g t h e p r o p e p t i d e s a n d

t h e t e l o p e p t id e s c a p t h e e n d s o f t h e D N A c o n s t r u c t s ,

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

e n d o n l y . T h e

SpeI

si te was used as the A si te (Fig. 2) .

A s d e s i g n e d , t h e p r o 0 0 ( I I ) D N A c a s se t t e sy s t e m c a n

b e u s e d t o a s s e m b l e D N A c o n s t r u c t s e n c o d i n g n o v e l

p r o c o l l a g e n s I I in w h i c h i n d i v i d u a l D - p e r i o d s o f t h e

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

s i n ce a n y D - p e r i o d c a s se t t e f r o m P l a sm i d I c a n b e a d d e d

Table II. Constructs assembled using the DN A cassette system.

Nam e Description Com position

FL full-length N -D 1-D2-D3-D4-Do.4-Ct

-D1 mis sing D1 N t - D 2 - D 3 - D 4 - D 0 . 4 - C

-D 2 missing D2 Nt-D1-D3-D4-Do.a-Ct

-D 3 m i s s in g D 3 N c D 1 - D 2 - D 4 - D o . 4 - C t

-D4 missing D4 Nt -DI-D2-D3-Do.4-C


8/12

1 1 2 W . V . A r n o l d e t a l.

Table I I I . Assem bly of funct ional proa l ( I I ) D NA cons t ruc t s us ing the DN A casse t te sys tem.

Func t ional Con s t ruc t Ligat ion React ions of Cloning Steps a

N t - D 1 - D 2 - D 3 - D 4 - D 0 . 4 - C t

(full length)

N , - D 2 - D 3 - D 4 - D o . 4 - C t

(missing D,)

N t - D 1 - D 3 - D 4 - D o . 4 - C t

m i s s i n g D 2 )

N t - D 1 - D 2 - D 4 - D 0 4 - C ~

(missing D3)

N t - D j - D 2 - D 3 - D 0 . 4 - C ~

m i s s i n g D 4 )

(1) Dl(p) + D 2 r ) = D 1 - D 2

(1) D3(p) + D 4 r ) = D 3 - D 4

1 ) D0.4(p) + Ct(r) = D 0 . 4 - C t

(2) Nt(p) + DI+D2(r) = N~-D1-Dz

(2) D3 +D4(p ) + Do.4+Ct(r = D 3 - D 4 - D o . 4 - C t

(3) N~ -Dt-D2(p) + D 3 - D 4 - D o . 4 - C t r ) = N t - D 1 - D 2 - D 3 - D 4 - D o . 4 - C t

(1) N~(p) + D 2 r ) = N ~ - D 2

(1) D3(p) + D 4 r ) = D 3 - D 4

(1) D 0.4(P) + C t(r) = D 0 . 4 - C t

(2) D3-D4(p) + D 0 . 4 - C t r ) = D 3 - D 4 - D 0 . 4 - C t

(3) N,-D2(p) + D3-D4-Do.4-Ct(r)= N t - D 2 - D 3 - D 4 - D o . 4 - C t

4 ) N~-D2-D3-D4-D04 -Cr(p) + Ct(r ) = * N t - D 2 - D 3 - D 4 - D o . 4 - C t

(1) Ndp) + Dl(r) = N~-D1

(1) D3(p) + Dr(r) = D3-D4

(1) D o.4(p) + C ~(r)

= D o . 4 - C t

(2) D3-D 4(p) + Do.4-C~(r) = D3-D4-Do.4-C~

(3) N~-D t(p) + D3-D4-D o.4-Cdr) = N~-D1-D3-D4-Do.4-C~

(1) DI(p) + D2(r) = D 1 - D 2

(1) D 4(p) + Do.4(r) = D4-Do.

(2) Ndp ) + D1-D2(r) = N,-D1-D2

(2) D4-D0.4(p) + Ct(r ) = D 4 - D o . 4 - C t

(3) N~-D1-D2(p) + D4-Do.4-C~(r)= N t - D 1 - D 2 - D 4 - D o . 4 - C t

(1) DI(p) + D2(r) = D 1 - D 2

(1) D3(p) + D0.4(r = D3-Do.

(2) N,(p) + D1-D2(r} = NcD 1-D 2

(2) D3-Do.4(p) + C t(r ) = D3-Do.4-C~

(3) Nt-D 1-D 2(p) + D3-Do.4-C~(r) = N~-D1-D2-D3-Do.4-C~

Th e (p) or (r) fol lowin g each casset te in the l igat ion react ion s indicates wh ethe r tha t cassette w as used as a p- or r-cassette, respec-

t ive ly, in the g iven c loning procedu re . Th e pr oal ( I I ) inser t in each p-casse tte , wi th o ne exce pt ion no ted in b , was re leased in tac t by

diges t ion wi th S p eI a n d BsrBI ( in the case of casset te inserts ending with D1, D2 o r D0.4); or S p e l a n d Bs tUI ( in the c ase o f cassette

inserts end ing D 3 o r D 4 } . The r -casse t te was o pened , wi th exc ept ion descr ibed in b , by diges t ion wi th S p eI a nd Srfl. The num be r i n

parentheses preceding each l iga t ion reac t ion descr ibes the t empora l order in which these reac t ions were per formed. Note tha t

many reac t ions were per formed concomi tant ly . The cons t ruc t s appear ing as a resul t of f ina l l iga t ion reac t ions were the funct ional

c ons t r uc t s w h i c h w e r e t r a ns fe r r e d t o a m a m m a l i a n e xp r e s s i on ve c t o r a nd us e d i n s ubs e que n t t r a ns f ec t i on p r oc e dur e s .

i n a n y d e s i r e d o r d e r t o t h e 5 ' e n d o f a C t c a s s e t t e i n P l a s -

m i d I I ( F ig . 3 ). T o r e d u c e t h e n u m b e r o f c l o n i n g s t e p s ,

c o m b i n e d c a s s e tt e s o f m o r e t h a n o n e D - p e r i o d w e r e

u s e d a s i n s e rt s ( T a b l e II I) . T h e f i n al D N A c o n s t r u c t w a s

c a p p e d b y t h e a d d i t i o n o f th e N t c a s s et t e t o th e 5 ' e n d o f

a n r - c a s s e t te .

S t a b l e t r a n s fe c t i o n o f H T 1 0 8 0 c e ll s w i t h f u n c t i o n a l

p r o c o l l a g e n I I c o n s t r u c t s

F i v e c o n s t r u c t s w e r e a s s e m b l e d f r o m t h e c a s s e t te s ( T a -

b l es I I a n d I I I) , a n d t h e s e q u e n c e s s p a n n i n g t h e j u n c t i o n s

w e r e a n a l y z e d b y d id e o x y n u c l e o t i d e s e q u e n ci n g . N o

m u t a t i o n s w e r e f o u n d i n t h e j u n c ti o n s ( n o t s h o w n ) . T h e

f iv e D N A c o n s t r u c t s w e r e e a c h p la c e d u n d e r t h e C M V

p r o m o t e r i n a m a m m a l i a n e x p r e s si o n v e c t o r c o n t a i n i n g

a g e n e e n c o d i n g n e o m y c i n r e s i s ta n c e ( F ig . 4 c ) a n d u s e d

f o r th e t r a n s f e c t i o n o f H T - 1 0 8 0 c e ll s. F o r e a c h c o n -

s t ru c t , a p p r o x i m a t e l y o n e h u n d r e d G 4 1 8 - r e s i s t a n t

c l o n e s w e r e s c r e e n e d b y W e s t e r n b l o t t i n g f o r th e s e c r e -

t i o n o f r e c o m b i n a n t p r o c o l l a g e n ( F i g. 5 ). T h e p e r c e n t a g e

o f p o s it i v e c lo n e s o b t a i n e d p e r t r a n s f e c t i o n w i t h e a c h o f

t h e f iv e D N A c o n s t r u c t s r a n g e d f r o m 1 0 t o 2 5 . T h e

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


9/12

B

+ +

A c D N A C a sse tt e S y s t e m 1 1 3

Q r~ a a a L L

I I I I I

m

typ

D1 pro

Figure 5. Screening of G 418-resistant clones by W estern blot-

ting. Pane l A: W estern blot demonstrating a positive signal in

one of four clones transfected with a recombinant p rocollagen

II m issing the Dl-perio d. The band b elow the recombinant

protein is a partially processed procd(II) chain lackin g a Dl-pe-

riod. Pan el B: A corresponding phosphor storage imag e of the

W estern blot demonstrating ~4C-radiolabeled proteins. Sy m-

bols: FN, fibronectin; Type IV, collagen IV; + , lane contain-

ing a positive clone.

o n t h e a m o u n t o f r e c o m b in a n t p r o t e in s e c re t i o n re l a ti v e

to e n d o g e n o u s t y p e I V c o ll a g e n s e c re t i o n a s d e t e r min e d

by SDS-PAGE fo l lowed by v isua l iza t ion of the rad io la -

b e l e d p r o t e in s w i th a p h o sp h o r s t o r a g e ima g e r ( F e r t a l a

e t a l. , 1 9 9 4 ) . T h e r a t i o o f s e cr e te d r e c o m b in a n t p r o t e in

to t y p e I V w a s e s se n t i a l l y t h e s a me in h ig h p r o d u c in g

c lo n e s t r a n s f e c t e d w i th c o n s t r u c t s l a c k in g a D p e r io d

a n d th e f u l l - l e n g th c o n s t r u c t . T h e r e f o r e , t h e re w a s n o i n -

d i c a t i o n o f i n t r a c e ll u l a r r e t e n t i o n o f t h e p r o t e in s.

P u r i f ic a t io n a n d a n a lys i s o f r e c o m b in a n t

procol lagens I I

C lo n e s e x p r e s s in g t h e r e c o m b in a n t p r o t e in s w e r e c h o -

se n f o r l ar g e s c al e p r o c o l l a g e n p r o d u c t io n , a n d t h e p r o -

c o l l a g e n s w e r e p u r i f i e d c h r o ma to g r a p h ic a l l y . E a c h r e -

c o mb in a n t p r o c o l l a g e n w a s c o n c e n t r a t e d ; S D S - P A G E o f

th e f i v e r e c o mb in a n t p r o c o l l a g e n s I I d e mo n s t r a t e d t h e y

were homogeneous (F ig . 6 ) . As expec ted , the procol la -

g e n s w i th a d e l e t e d D - p e r io d ( 2 3 4 a min o a c id s ) mi -

g r a t e d m o r e r a p id ly t h a n t h e w i ld - ty p e p r o t e in . T h e i n i-

t i a l y ie ld s o f t h e r e c o mb in a n t p r o c o l l a g e n s f r o m 4 1 o f

c u l t u r e me d iu m w e r e a b o u t 0 . 5 t o 1 . 0 mg . H o w e v e r ,

la rge and va r iab le losses were encounte red in the f ina l

p r o a l I )

p r o c 2 I )

Figure 6. Purified recombinan t proco llagens II. SDS-PAGE of

recomb inant procollagens II missing D~, D2, D a n d D 4 periods.

Electrophoresis was performed under reducing conditions

using 7.5% polyacrylamide ge l. Sym bols : -D1 , -D2 , etc.,

procd(II) chains in wh ich specific D-periods w ere deleted; I

pro, standard of wild-type procollagen I; FL, recombinant

full-length procollagen II (Fertala et al., 19 94).

c o n c e n t r a t i o n s t e p r e q u i r e d f o r C D a n a ly s i s , a n d t h e

f ina l y ields were 39 to 384 lag .

E a c h p r o c o l l a g e n w a s a n a ly z e d f o r i t s a min o a c id

c o mp o s i t i o n ( n o t sh o w n ) . T h e o b se r v e d v a lu e s a g r e e d

w e l l w i th t h e c a l c u l a t ed v a lu e s f o r v a r i a n t s o f p r o c o l l a -

gen lack ing spec i f ic D-pe r iods , wi th three except ions :

t h e v a lu e s f o r g lu t a m a te p lu s g lu t a min e ( G lx) w e r e c o n -

s i s t en t l y l o w e r t h a n c a l c u l a t e d ( 7 0 t o 7 6 o b se r v e d vs 96

to 104 ca lcu la ted) , the va lues for h is t id ine were cons is -

ten t ly h ighe r (11 to 31 vs 7 a n d 8 c a l c u l a t e d ) , a n d t h e

v a lu e s f o r t y r o s in e w e r e a l so c o n s i s t e n t l y h ig h e r t h a n

ca lcu la ted (19 to 40 vs 7 and 8 ca lcu la ted) . The reasons

f o r t h e t h r e e e x c e p t io n s w e r e n o t a p p a r e n t . E s se n t i a l l y

th e s a me v a lu e s w e r e o b t a in e d w i th d u p l i c a t e s a mp le s

a n d w i th a l l f o u r d i f f e r e n t r e c o mb in a n t p r o c o l l a g e n s .

There fore , the except iona l va lues were un l ike ly to be ex-

p l a in e d b y c o n t a min a n t s . H ig h e r v a lu e s f o r h i s t i d in e ( 8

vs 2 c a l c u l a t e d ) a n d t y r o s in e 9 vs 2 ca lcu la ted) were

previous ly obse rved for fu l l - length type I I p rocol lagen

f r o m th e s a me r e c o mb in a n t sy s t e m ( F e rt a l a e t al ., 1 9 9 4 ) .

C o n fo rm a t io n o f t h e r e c o m b in a n t p ro c o lla g en s

T o a s se s s t h e c o n f o r ma t io n o f t h e r e c o mb in a n t p r o -

t e in s , t h e r e c o mb in a n t p r o c o l l a g e n s w e r e e x a min e d b y

C D . T h e sp e c t r a b e tw e e n 2 0 5 a n d 2 6 0 n m w e r e s imi l a r

to the cha rac te r i s t ic spec t rum of t r ip le -he l ica l type I I


10/12

114 W.V. Arn old e t a l .

p r o c o l l a g e n ( n o t sh o w n ) . T h e ma g n i tu d e o f C D s ig n a l a t

t h e m a x i m u m a r o u n d 2 2 1 n m o f t h e r e c o m b i n a n t p r o -

t e in s w a s s l i g h t ly l o w e r t h a n t h e ma x im u m o f t h e

w i ld - ty p e p r o c o l l a g e n , a n o b se r v a t i o n c o n s i s t e n t w i th

the lower amount of t r ip le he l ica l r e s idues present in the

r e c o mb in a n t p r o t e in s . F o r r e a so n s t h a t w e r e n o t a p p a r -

e n t , t h e sh a p e o f t h e sp e c t r u m w a s s l i g h tl y d i f f e r e n t w i th

th e D 3 p r o c o ll a g e n , a n d t h e m a g n i t u d e o f th e m a x i m u m

w a s t h a t o f t h e w i ld - t y p e p r o c o l l a g e n . I n f u r th e r e x p e r i -

me n t s , a l l t h e r e c o mb in a n t p r o t e in s w e r e sh o w n to u n -

d e r g o sh a r p t h e r ma l t r a n s i t i o n s s imi l a r t o t h o se o f n a -

t ive type I I co l lagen (A rnold e t a l . , in prepa ra t ion ) .

iscussion

T h e D N A c a s set t e sy s te m d e sc r ib e d h e r e t o g e th e r w i th

th e p r e v io u s ly d e sc r ib ed e x p r e s s io n sy s t e m f o r r e c o mb i -

nant procol lagens (Fe r ta la e t a l . , 1994) can be used to

prepa re nove l f ib r i l la r p rocol lage ns . The f ide l i ty of the

over -a l l sys tem in prepa r ing these nove l pro te ins was

v e r i fi e d b y p a rt i a l D N A se q u e n c in g , b y a min o a c id a n a l -

y s i s o f t h e p r o t e in s , a n d b y c o n f o r ma t io n a l a n a ly s i s b y

C D . T h e D - p e r io d d e l e t e d p r o c o l l a g e n s I I w e r e f o ld e d

in to the t r ip le he l ica l s t ruc ture a s show n by the C D spec -

t r u m. T h e r e c o m b in a n t mo le c u l e s a r e t h e r e fo r e u se f u l t o

d e t e r min e t h e c o n t r i b u t i o n o f d i f f e r e n t r e g io n s t o t h e r -

ma l s t a b i l i t y (E n g e l, 1 9 8 7 ; D 6 1 z a n d H e id e m a n n , 1 9 8 8 ;

Kadle r e t a l . , 1988; Mor r is e t a l . , 1990; Weste rhausen e t

a l . , 1990; B / ich inge r and Davis , 1991; B / ich inge r e t a l . ,

1993; Fe r ta la e t a l . , 1993) , fo ld ing , f ib r i l fo rma t ion

( P r o c k o p a n d H u lme s , 1 9 9 4 ) a n d sp e c i f i c i n t e r a c t i o n s

w i th o th e r mo le c u l e s .

T h e f l e x ib i l i ty o f t h e D N A c a s set t e sy s t e m a l l o w s f o r

th e c r e a t i o n o f a n u mb e r o f i n f o r ma t iv e p r o c o l l a g e n I I

D N A c o n s t r u c t s . I t sh o u ld b e n o t e d t h a t t h e D - p e r io d

div is ion of the t r ip le -he l ica l r eg ion of pro ix l ( I I ) was a

so me w h a t a r b i t r a r y d iv i s io n o f t h e f u n c t i o n a l d o ma in s

o f th e p r o t e in ( H u lm e s e t a l ., 1 9 7 3 ; C h a p m a n , 1 9 8 4 ).

Smal le r or la rge r casse t te s can readi ly be synthes ized .

T h e n u c l e o t i d e s e q u e n ce c o d in g f o r t h e h u m a n p r o 0c l (I I)

t r ip le -he l ica l r eg ion conta ins 151 sepa ra te loca t ions

w h e r e a r f l s i te m ay be enginee red a s the 5 end o f a

casse t te . The 3 end of a casse t te can be te rmin a ted in

any codon for a pro l ine , a lan ine or se r ine . S imi la r cas-

se t te s can a lso be made for the propept ides . Fur the r -

mo r e , c o mp le x c o n s t r u c t s c a n n o w b e a s se mb le d f r o m

th e i n t e r me d ia t e c a s se t t e c o n s t r u c t s a l r e a d y a v a i l a b l e

(Table I I I ) . Also , the DNA casse t te sys tem can eas i ly be

a d a p t e d t o o th e r p r o c o l l a g e n c D N A s a n d e v e n t o

c D N A s f o r o th e r l a r g e p r o t e in s . T h e c r u x o f t h e s t r a te g y

is to in t roduce appropr ia te b lunt -cu t t ing re s t r ic t ion s i te s

w h ic h a r e c o mp a t ib l e w i th t h e n a t i v e n u c l e o t i d e s e -

quence encoding the pro te in of in te res t . This i s gene ra l ly

not d i f f icu l t , g iven the ava i lab i l i ty of a la rge number of

b lu n t - c u t t i n g r e s tr i c t io n e n z y me s a n d t h e n e e d t o ma tc h

only one -ha l f o f the re s t r ic t ion s i te to the na t ive pro te in

nuc leo t ide sequence .

O n e o f t h e u n e x p e c t e d f i n d in g s h e r e w a s t h a t f o u r

mo d i f i e d p r o c o l l a g e n I I mo n o me r s l a c k in g sp e c i f i c

D - p e r io d s w e r e e a c h s e c r e te d b y t h e m a m ma l i a n e x p r e s-

s ion sys tem as t r ip le -he l ica l monomers a t 37 C . P rev i -

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

r e p l a cin g a c o d o n f o r a n o b l ig a t e g ly cin e i n p r o c o l l a g e n

I l o w e r t h e me l t i n g t e mp e r a tu r e b e lo w 3 7 C a n d c a u se

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

( W e s t e r h a u se n e t a l . , 1 9 9 0 ; P r o c k o p a n d K iv i r i k k o ,

1995; K uivan iemi e t a l . , 1996). A lso , an in- f ram e dele -

t i o n o f t h r e e e x o n s a n d 8 4 a m in o a c id s i n t h e p r o 0 d ( I )

cha in of type I p rocol lagen had a s imi la r e f fec t (Kuiv-

aniemi e t a l . , 1996) . The obse rva t ion he re tha t de le t ion

o f a c o mp le t e D - p e r io d o f 2 3 4 a min o a c id s d id n o t p r e -

v e n t mo n o m e r s o f p r o c o l l a g e n I I f r o m f o ld in g in to a n a -

t i v e c o n f o r ma t io n su p p o r t s a l a r g e b o d y o f i n f o r ma t io n

su g g e s t in g t h a t e a c h D - p e r io d c o n t a in s so me a m in o a c id

sequences tha t s tab i l ize and some tha t do not s tab i l ize

th e t r i p le h e l i x ( E n g e l, 1 9 8 7 ; D 6 1 z a n d H e id e ma n n ,

1988; Kadle r e t a l . , 1988; Mor r is e t a l . , 1990; Weste r -

hausen e t a l . , 1990; B~ichinge r and Davis , 1991;

B~ichinger e t a l . , 19 93; Fer ta l a e t a l . , 19 93) .

S ince the modif ied procol lagen I I monomers were se -

c re ted in a t r ip le -he l ica l conformat ion , the re su l t s sug-

ges t tha t the s t r a tegy of D-pe r iod casse t te s can be used

to map spec i f ic b inding s i te s on f ibr i l la r co l lagens . Ob-

se r v a t io n s o n t h e p a t t e r n s o f m o n o me r a s se mb ly

i n v i t ro

a n d o n t h e p a t t e r n s o f c o v a l e n t c r o s s- l in k s f o u n d w i th in

mature f ibr i l s sugges ted (Chapman, 1984; P iez , 1984;

S i lve r e t a l . , 1992) but d id not prove (Pa rk inson e t a l . ,

1 9 9 4 ) t h a t t h e m o n o m e r s a s se mb le i n f ib r il s b e c au se o f

spec i f ic b inding in te rac t ions tha t occur be tween s i te s in

the t r ip le -he l ica l domains and s i te s in the shor t te lopep-

t i d e s w i th n o n - h e l i c a l s eq u e n c e s f o u n d a t e a c h e n d o f t h e

monomers . Also , a se r ie s of obse rva t ions sugges ted tha t

a s t h e mo n o m e r s a s se mb le i n to f i b r il s a n d a f t e r t h e f i b-

r i ls a re form ed, the co l lagens u nde rgo a se ries of spec i fic

b in d in g i n t e r a c t i o n s w i th o th e r ma c r o mo le c u l e s o f t h e

e x t r a c e l l u l a r ma t r i x ( H e d b o m a n d H e in e g f i r d , 1 9 9 3 ;

San Antonio e t a l . , 1994) . In addi t ion , i t has been

demonst ra ted tha t co l lagen f ibr i l s se rve a s subs t ra te s for

ce ll b inding , cel l spreading a nd ce l l s igna l ing thro ug h in-

tegr ins and o the r r eceptor s (S taa tz e t a l . , 1991; Gulbe rg

e t a l . , 1992; Weston e t a l . , 1994) . However , de f in i t ive


11/12

A c D N A C a s s e tt e S y s te m 1 1 5

d a t a o n s p e c i f ic b i n d i n g s it e s a r e n o t a v a i l a b le , p r i m a r i l y

b e c a u s e s i t e - d ir e c t e d m u t a g e n e s i s o f t h e w h o l e

m o n o m e r s is t e c h n i c a l ly d i f fi c u lt , a n d m o s t s h o r t p e p -

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

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

t i o n s. T h e s t r a t e g y o f D - p e r i o d c a s s e tt e s s h o u l d m a k e i t

p o s s i b l e t o o v e r c o m e t h e s e p r o b l e m s .

cknowLedgements

T h i s w o r k w a s s u p p o r t e d i n p a r t b y N a t i o n a l I n s t it u te s o f

H e a l t h G r a n t A R - 3 9 7 4 0 , a g r a n t f r o m t h e L u c i l l e P . M a r k e y

C h a r i t a b l e T r u s t , a n d a g r a n t f r o m t h e S h r i n e r s Ho s p i t a l .

eferences

A l a - K o k k o , L . a n d P r o c k o p , D . J. : C o m p l e t i o n o f t h e i n t ro n -

e x o n s t r u c t u r e o f th e g e n e f o r h u ma n t y p e I I p r o c o l l a g e n

C O L2 A 1 ) . V a r i a t i o n s i n t h e n u c l e o t i d e s e q u e n c e s o f t h e a l l e-

l es f r o m t h r e e c h r o m o s o m e s . G e nom i c s 8 4 5 4 - 4 6 0 , 1990.

Al a - K o k k o , L . , Hy l a n d , J . , S mi t h , C . , K i v i r i k k o , K . L , J i me n e z ,

S . A . a n d P r o c k o p , D . J . : Ex p r e s s i o n o f a h u ma n c a r t i l a g e p r o -

c o l l a g e n g e n e C O L2 A 1 ) i n mo u s e 3 T3 c el ls . J. Biol . Chem.

266: 1 4 1 7 5 - 1 4 1 7 8 , 1991.

B/ ich inger , H.P . and D av i s , J .M. : Se quence spec i f i c the rm a l s t a -

b i l i ty o f the co l l agen t r ip le he l ix . Int . J. Biol . Macromol. 13:

1 5 2 - 1 6 2 , 1991 .

B / ic h in g er , H . P . , M o r r i s , N . P . a n d Da v i s , J .M . : T h e r m a l s t a b i l i ty

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

t a t i o n s i n o s t e o g e n e s i s i mp e r f e c t a o n t h e t r i p le h e l i x o f t y p e I

co l l agen . Am . J . M e d . G e ne t . 45: 1 5 2 - 1 6 2 , 1993.

B a l d wi n , C . T . , R e g i n a t o , A . M . , S mi t h , C . , J i me n e z , S . A . a n d

P r o c k o p , D . J. : S t ru c t u re o f c D N A c l o ne s c o d in g f o r h u m a n

t y p e I I p r o c o l l a g e n . Th e ~ 1 I I ) c h a in i s mo r e s i mi l a r t o t h e

c x l I ) c h a i n t h a n t wo o t h e r a c h a i n s o f f i b r i l la r c o l l a g e n s.

Bi oc he m . J. 262 : 521 - 5 28 , 1989 .

C h a p ma n , J . A . : M o l e c u l a r o r g a n i z a t i o n i n t h e c o l l a g e n f i b r i l .

In : Connect ive Ti ssue Matr ix e d . b y Hu k i n s , D . W . L . , Ve r l a g

C h e mi e , De e r f i e l d B e a c h , F L , 1 9 8 4 , p p . 8 9 - 1 3 2 .

D6 1 z , R . a n d He i d e ma n n , E . : I n f l u e n c e o f d i f f e r e n t t r i p e p t i d e s

on the s t ab i l i ty o f the co l l agen t r ip le he l ix . I . Ana lys i s o f the

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

Bi opo l y m e r s 25 : 1 0 6 9 - 1 0 8 0 , 1986 .

En g e l , J . , C h e n , H . - T . , P r o c k o p , D . J . a n d K l u mp , H . : Th e t r i p le

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

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

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

L - P r o - L - P r o -G l y ) , a n d L - P r o - L - H y p - G l y )n . B iopo lymers 16 :

6 0 1 - 6 2 2 , 1 9 7 7 .

Enge l , J . : Fo ld ing an d u n fo ld ing o f co l l agen t r ip le he l ices . In :

Advances in Meat Research, e d . b y P e ar s o n , A . M . , D u d s o n ,

T . R . a n d B a i l e y , A . J . , Va n No s t r a n d R e i n h o l d , 1 9 8 7 , p p .

1 4 5 - 1 6 1 .

F e r t a l a , A . , S i e r o n , A . L . , G a n g u l y , A . , L i , S .- W . , A l a - Ko k k o , L . ,

A n u m u l a , K . R . a n d P r o c k o p , D . J . : S y nt h es is o f r e c o m b i n a n t

h u m a n p r o c o l l a g e n I I in a s t a b l y t ra n s f e c t e d t u m o r c e l l l in e

H T - 1 0 8 0 ) . Biochem. J . 298: 3 1 - 3 7 , 1 9 9 4 .

Ge r m a n n , H . -P . a n d He i d e m a n n , E . : A s y n t h e t i c mo d e l o f c o l l a -

g e n : a n e x p e r i m e n t a l i n v e s t i g a t io n o f t h e t r i p l e - h e l ix s t a b i l it y .

Bi opo l y m e r s 27 : 1 5 7 - 1 6 3 , 1 9 8 8 .

Gu l l b e r g , D . , Ge h l s o n , K . R . , Tu r n e r , D . C . , Ah l e n , K . , Z i j e n a h ,

L.S . , Ba rnes , M.J . and Rub in , K. : Ana lys i s o f 0 t1~1 , a2131

and c t3131 in teg r ins in ce l l - co l l agen in te rac t ions : iden t i f i ca t ion

o f c o n f o r ma t i o n d e p e n d e n t a 1 1 3 1 b i n d i n g s i t e s i n c o l l a g e n I .

E M B O J . I 1: 3 8 6 5 - 3 8 7 3 , 1 9 9 2 .

H e d b o m , E . a n d H e i n e g fi r d, D . : B i n d in g o f f i b r o m o d u l i n a n d

decor in to sepa ra te s i t e s on f ib r i l l a r co l l agens . J. Biol . Chem.

268: 2 7 3 0 7 - 2 7 3 1 2 , 1 9 9 3 .

He i d e ma n n , E . a n d R o t h , W . : S y n t h e s i s a n d i n v e s t i g a t i o n o f

c o l l a g e n mo d e l p e p t i d e s . Adv . P o l y m . S c i . 43 : 1 4 3 - 1 5 3 ,

1983 .

Hu lmes , D.J .S . , Mi l l e r , A. , Pa r ry , D.A.D, P iez , K.A. and Wood-

h e a d - G a l l o wa y , J . : An a l y s is o f th e p r i m a r y s t r u c t u r e o f c o l la -

g e n f o r t h e o r i g i n s o f mo l e c u l a r p a c k i n g . J. Mol. Biol . 79:

1 3 7 - 1 4 8 , 1 9 7 3 .

I n o u y e , K . , Ko b a y a s h i , Y . , Ky o g o k u , Y . , K i s h i d a , Y . , S a k a k -

i b a r a , S . a n d P r o c k o p , D . J .: S y n t h e s is a n d p h y s i c a l p r o p e r t i e s

o f h y d r o x y p r o l i n e - p r o l i n e - g l y c i n e ) 1 0 : Hy d r o x y p r o l i n e i n

t h e X- p o s i t i o n d e c r e a s e s th e m e l t i n g te m p e r a t u r e o f t h e c o ll a -

gen t r ip le he l ix . Arch . Bi oc he m . B i ophy s . 219 : 1 9 8 - 2 0 3 ,

1982 .

Ka d l e r , K . E . , Ho j i ma , Y . a n d P r o c k o p , D . J . : As s e mb l y o f t y p e I

c o l l a g e n d e n o v o . B e t we e n 3 7 a n d 4 1 C t h e p r o c e s s i s l i m-

i t e d b y m i c r o - u n f o l d i n g o f m o n o m e r s . J . Biol . Chem. 263:

1 0 5 1 7 - 1 0 5 2 3 , 1 9 8 8 .

Ki ihn , K . , F ie t zek , P. and Ki . ihn , J . : The a c t ion o f p ro teo ly t i c

e n z y m e s o n c o l l a g e n . Bi oc he m . Z . 344 : 4 1 8 - 4 3 4 , 1 9 6 6 .

K u i v a n i e m i , H . , T r o m p , G . a n d P r o c k o p , D . J . : M u t a t i o n s i n

f ib r i l l a r co l l agen types 1, I I , I I I and XI ) , f ib r i l - a s soc ia ted co l -

l a g e n t y p e I X) , a n d n e t w o r k - f o r m i n g c o l l a g e n t y p e X) c a u s e

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

H u m a n M ut a t i on 9 : 300 - 315 , 1997 .

M orr i s , N .P ., Wa t t , S .L., Dav i s , J .M . and B / i chinger , H.P .: Un-

f o l d i n g i n t e r me d i a t e s i n t h e t r i p l e h e l i x t o c o i l t r a n s i t i o n o f

b o v i n e ty p e X I c o l la g e n a n d h u m a n t y p e V c o ll a g en s a 1 2 ) a 2

and 0d~21x3 . J. Biol . Chem. 265: 1 0 0 8 1 - 1 0 0 8 7 , 1 9 9 0 .

P a r k i n s o n , J . , Ka d l e r , K . E . a n d B a s s, A . : S e l f- a s s e mb l y o f

r o d - l i k e p a r t ic l e s i n t wo d i me n s i o n s : a s i mp l e mo d e l o f c o l l a -

gen f ib r i l logenes i s . Physical Rev. E 50: 2 9 6 3 - 2 9 6 6 , 1 9 9 4 .

P i ez , K . A . : M o l e c u l a r a n d a g g r e g a t e s t r u c t u r e s o f t h e c o l l a g e n s .

In: Extracellular Matrix Biochemistry, ed . by P iez , K.A. and

R e d d i , A . H . , E l s e v i e r , Ne w Yo r k , 1 9 8 4 , p p . 1 - 4 0 .

P r i v a lo v , EL . : S t a b i l i ty o f p r o t e i n s . P r o t e i n s wh i c h d o n o t p r e -

s e n t a s i n g l e c o o p e r a t i v e s y s t e m. Adv. Prote ins Chem. 35:

1 - 1 0 4 , 1 9 8 2

P r o c k o p , D . J . a n d H u l m e s , D . J .S . : As s e m b l y o f c o l l a g e n f ib r i ls

de nov o f r o m s o l u b l e p r e c u r s o r s : P o l y me r i z a t i o n a n d c o p o l y -

me r i z a t i o n o f p r o c o l l a g e n , p N- c o l l a g e n , a n d mu t a t e d c o l l a -

gens . In : Extracel lu lar Matr ix A ssem bly an d St ructure , e d . b y

Yu r c h e n c o , ED. , B i r k , D . E . a n d M e c h a m, R . P . , Ac a d e mi c

P r e s s , Ne w Yo r k , 1 9 9 4 , p p . 4 7 - 9 0 .

P r o c k o p , D . J . a n d K i v i r i k k o , K . I . : C o l l a g e n s : mo l e c u l a r b i o l -

ogy , d i seases , and po ten t i a l s fo r the rapy . Annu . Re v .

Biochem. 64: 4 0 3 - 4 3 4 , 1 9 9 5 .

P r o c k o p , D . J . , B e r g, R . A . , K i v i r i k k o , K . I . a n d U i t t o , J . : I n t ra c e l -

lu la r s t eps in the b iosyn the s i s o f co l l agen . In : Bi oc he m i s t r y o f

Collagen, e d . b y R a m a c h a n d r a n , G . N . a n d R e d d i , A . H . ,

P l e n u m , N e w Y o r k , 1 9 7 6 , .p p . 1 6 3 - 2 7 3 .

R o t h , W . a n d He i d e m a n n , E . : T r i p l e h e li x - c o il t r a n s i ti o n o f c o -

v a l e n t l y b r i d g e d c o l l a g e n - l i k e p e p t i d e s . Bi opo l y m e r s 19 :

1 9 0 9 - 1 9 1 7 , 1 9 8 0 .

R y h ~ i n e n , L . , Za r a g o z a , E . J . a n d U i t t o , J . : C o n f o r ma t i o n a l s t a -

b i l i t y o f t y p e I c o l l a g e n t r i p l e h e l i x : e v i d e n c e f o r t e mp o r a r y

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


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