In vitro reconstitution of wool-IF.pdf

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n v i t r o r e c o n s t i t u t i o n o f w o o l

i n t e r m e d i a t e f i l a m e n t s

Helga Thomas, Andrea Conrads,

K i m - H b P h a n ,

Monika van de Li icht and

H e l m u t

Z a h n t

D e u t s c h e s W o l l f o r s ch u n g s i n st i t u t a n d e r R W T H A a c h e n V e l t m a n p la t z 8 D - 5 1 0 0 A a c h e n F R G

(Received 7 February 1986; revised 7 May 1986)

Al thou yh the hard a-kerat ins o f woo l are recognized as mem bers oJ the in termediate j i lame nts by sequence

comparison thus far a l l a t t empts on recons t i tu t ion o f woo l a-kerat in f i lamen ts

in vitro

have f i~iled. Here we show

that ox idat ive sulphito lys is rather than the prev ious ly used S-carboxy methy lat ion i s the method Of choice to

prepare a -kerat in der ivatives sui table ., (or assemb ly exper ime nts . O nce the protec t ing S-sulpho grou p i s removed

by 2 - m e r c ap t oe thano l in vitro . fi lament form at ion can be induced. Elec t ron micrographs show f i laments wi th a

diameter o f ~ 1 1 nm as in a l l o ther in termediate . fi laments . Thu s , f i lament Jormation o/ c t -kerat ins does not

require the presence o f m atr ix prote ins .

Keywords: Intermediate filaments: in vitro reconstitu tion: negative staining; oxidative sulphitolysis

Introduction

I n t e r me d ia t e f i l a me n t s ( I F ) a r e a c o n s t i t u e n t o f t h e

c y to s k e l e to n p r e s e n t i n a lmo s t a l l v e r t e b r a t e c e l l s . T h e y

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

l e v e l a s f i l a me n to u s s t r u c tu r e s w i th a c h a r a c t e r i s t i c

d i a m e t e r o f 7 - 1 n m w h i c h is in t e r m e d i a t e b e t w e e n th a t

o f th e a c t i n - c o n t a i n i n g m i c r o f i l a m e n t s ( 5 -7 n m ) a n d t h e

t u b u l i n - c o n t a i n i n g m i c r o t u b u l e s (2 5 n m ) 1. A l t h o u g h I F

a r e a g r o u p o f s t r u c tu r a l l y s imi l a r f il a me n t s s h o w in g

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

h e l ic a l c o n f o r m a t io n , t h e y c a n b e d iv id e d i n to f iv e d i s t i n c t

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

c r i t e r i a : k e r a t i n s , v ime n t in , d e s min , g l i a l f i l a me n t s a n d

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

th e k e r a t i n s r e p r e s e n t t h e mo s t c o m p le x c l a s s 6 . R e c e n t l y ,

the mic ro f ibr i l s o f ha r d c~-ke ra tins hav e been inc lude d in to

th e c l as s o f I F b a s e d o n t h e i r s e q u e n c e a n d s t r u c tu r a l

h o m o lo g i e s w i th t h e o th e r I F p r o t e in s 4 8.

G e n e r a l l y , a m in o a c id se q u e n c e d a t a h a v e r e v e a l e d

t h a t I F s u b u n i t s a r e c o m p o s e d o f a c e n t r a l s -h e l ic a l

d o m a i n o f c o n s e r v e d l e n g th a n d s e c o n d a r y s t r u c t u re a n d

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

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

v a r i a t i o n s i n s i z e a n d p r im a r y s t r u c tu r e 5 '9 t l

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

o f I F h a v e b e e n o b s e r v e d i n t h e e le c t r o n mic r o s c o p e a f t e r

me c h a n i c a l d i s r u p tu r e o f w o o l f ib r e s l 2, i n t a c t h a i r k e r a t i n

f i l a me n t s h a v e b e e n i s o l a t e d o n ly f r o m f o l l i c l e s o f r a t

v ib r a s s a e a n d t h e i n n e r r o o t s h e a th o f g u in e a - p ig

h a i r t 3 't * . A t t e mp t s h a v e b e e n m a d e t o r e c o n s t i t u t e

mic r o f ib r i l l a r p r o t e in s f r o m h a r d k e r a t i n f i b r e s ( w o o l )

a f t e r d ig e s t i o n o f t h e n o n - h e l i c a l t a i ls tS ' t 6 , w h e r e a s i n t a c t

p r o t e in m a te r i a l f r o m th e o th e r I F - c o n t a in in g t i s s u e s, e. g .

* Partly presented at the EMBO-Workshop Intermediat e Filaments:

Structure, Function and Patho logy' , Irsee, 27-30 April 1985 as well as a

poster demonstrat ion at the 3rd Meeting of the European Cytoskeletal

Club, 'The Cytoskeleton in De velopment and Pathogenesis' , Bielefeld,

7-10 September 1985.

+ To whom correspondence should be addressed.

0141-8130/86/050258~7503.00

~ 1986 Butterworth & Co. [Publishers) Ltd

2 5 8 I n t . J . B io l . M a c r o mo l . , 1 9 86 , V o l 8 , O c to b e r

a s t r o c y t e s , s mo o th mu s c l e c e l l s , k e r a t i n i z in g a n d n o n -

k e r a t i n i z in g e p i t h e l i a h a v e b e e n i s o l a t e d a n d

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

in v i t ro 17.

T h e s e d i f f ic u l ti e s w i th h a r d k e r a t i n s a r e d u e t o t h e

e x t r e me in s o lu b i l i t y o f t h e w o o l mic r o f ib r i l l a r p r o t e in s ,

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

e x i s t e n c e o f a s u lp h u r - r i c h i n t e r mic r o f ib r i l l a r ma t r i x i n

w o o l a n d h a i r , l e a d in g t o a c o - e x t r a c t i o n o f b o th

c o mp o n e n t s a f t e r c l e a v a g e o f t h e c y s t i n e l i n k a g e s .

U p t il l n o w e x t r a c t i o n o f w o o l k e r a t i n s i n v o lv in g a

s e p a r a t i o n i n to mic r o f ib r i l l a r a n d m a t r i x p r o t e in s h a s

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

o x id a t i o n p r o c e d u r e s 1 8't 9. T h e s e l e d , h o w e v e r , t o n e w

in t r o d u c e d c h a r g e s . S o f a r n o

i n v i t r o

r e c o n s t i t u ti o n o f

w o o l mic r o f ib r i l s b a s e d o n t h e s e i s o l a t i o n me th o d s h a s

b e e n d e s c r i b e d . W i th a mo d i f i e d o x id a t i v e s u lp h i t o ly s i s

p r o c e d u r e a c c o r d i n g t o B a i l e y 2 ' 2 1 w e h a v e a l r e a d y

s h o w n 2 2-2 4 t h e p o s s ib i l i ty o f e x t r a c t i n g w o o l k e r a t i n s i n

th e S - s u lp h o f o r m, w h ic h a l l o w s a s e p a r a t i o n i n to

mic r o f ib r i l l a r a n d ma t r i x p r o t e in s a s w e l l a s t h e

r e f o r m a t io n o f c y s t i n e b ri d g e s . I t w a s t h e r e f o r e o b v io u s t o

u s e t hi s t e c h n i q u e in o r d e r t o a t t e m p t - - a s i n t h e c as e o f

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

f i l a me n t s w h ic h c a n b e c h a r a c t e r i z e d b y e l e c t r o n

mic r o s c o p y . W e s h o u ld l i k e t o d e s c r i b e h e r e t h e

e x t r a c t i o n p r o c e d u r e a s s u c h , i n v o lv in g s o lu b i l i z a t i o n

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

i n v i t r o

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

wool mic rof ibr i l s .

Mater ia l s and methods

S t a r t i n g m a t e r i a l

U n d a m a g e d L in c o ln w o o l (~b 3 8 / a m, w i th o u t t ip s ).

E x t e r n a l l i p id w a s r e mo v e d b y a S o x h l e t e x t r a c t i o n

p r o c e d u r e u s i ng d i c h l o r o m e t h a n e f o r 2 h.

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

O n e g r a m s n i p p et s o f L i n c o l n w o o l ( a p p r o x i m a t e l y

2 m m ) w e r e e x t r a ct e d w i t h 1 0 0 m l o f t h e f ol l o w in g

s u lp h i t o ly s i s r e a g e n t : 0 . 2 M N a 2 S O 3 , 0 . I M N a z S a O 6 i n


2/7

In v i t ro

r e c o n s t i t u t i o n o f w o o l i n t e r me d i a t e f i l a me n t s : H . T h o ma s

et al.

8

M

urea, 0 .1 MTri s , pH 9 .5 , fo r 24 h a t room temp era tu re .

The inso lub le res idue was removed af t e r cen t r i fuga t ion

( 4 0 0 0 r e v / mi n , r o o m t e mp e r a t u r e , 3 0 mi n ) a n d t h e

superna tan t was d ia lysed aga ins t de ion ized water fo r 2

days (Visking dialys is tube 27/32).

Separa t ion o f matr ix and micro f ibr i l lar pro te ins

After concen t ra t ing the S-su lpho-kera t e in -con ta in ing

so lu t ion to 100 ml , 2 ml o f a 1 M z inc ace ta t e so lu t ion w ere

added ( resu l t ing pH 6 .045 .5 ) and the p rec ip i t a t ed

pro te ins separa t ed by cen t r i fuga t ion (4000 rev /min , roo m

t e mp e r a t u r e , 3 0 mi n ) . T h e p e l l e t w a s s u s p e n d e d i n 1 %

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

approx imate ly 1% and d ia lysed aga ins t 30 - fo ld excess o f

d i s ti l led wa ter and 0 .05 M sod iu m te t rab ora te so lu t ion .

The p ro te ins were aga in p rec ip i t a t ed wi th z inc ace ta t e ,

red i s so lved in sod ium c i t ra t e , d i a lysed aga ins t d i s t i l l ed

wa ter a nd f ina lly f reeze-d r i ed .

In v i t ro

r e c o n s ti t u ti o n o f w o o l f i l a me n t s

The p rec ip i t a t ed f reeze-d r i ed p ro te ins were d i s so lved in

the fo l lowing so lu t ion (2 mg /ml ) an d kep t fo r 12-15 h a t

6C: 8 M urea , 8 % f l -m ercap toe thano l , 0 .05 M Tr i s -HC 1

pH 7 .5 . Af te r cen t r i fuga t ion (10 0000 g , 4C, 10m in)

f i lamen t s were recons t i tu t ed b y d ia lys ing the supern a tan t

a t 6 C o v e r n i g h t a g a i n s t 4 M u r e a , 1 0 mM T r i s - H Cl p H

7.5 , 25mM f l -mercap toe thano l . Fur ther d i a lys i s was

per fo rmed aga ins t 10mM Tr i s -HC1 pH 7 .5 , 10mM f l -

me r c a p t o e t h a n o l f o r a n o t h e r p e r i o d o f 1 2 - 2 4 h a t 6 C .

So lu t ions o f recon s t i tu t ed w ool kera t in f i lamen t s were

d i lu t ed to approx imate ly i mg/ml wi th the l as t d i a lys i s

buf fer and a d ro p o f th i s so lu t ion p laced on a g l as s p l a t e

covered wi th paraf i lm. A carbon coa ted g r id (Cu ,

3 .05 mm , 200 mesh T ed Pe l l a Inc .) was p laced w i th the

c a r b o n s i de o n t o t h e s u r f a c e o f th e d r o p , a l lo w i n g t h e

p r o t e i n s to a d s o r b d u r i n g 2 - 5 m i n . T h e s a mp l e s w e r e th e n

i mme d i a t e l y c o n t r a s t e d w i t h u r a n y l a c e t a t e ( 1 - 2 % ,

f i l t e red so lu t ion) dur ing 60 s and a i r d r i ed .

Elec tron microscopy

Neg at ive ly s ta ined spec im ens were examined in a Ze i s s

E M 1 0 9 e l e c t r o n mi c r o s c o p e t h a t w a s o p e r a t e d a t a n

acce le ra t ing vo l t age o f 50 kV. E lec t ron m icrograph s w ere

r e c o r d e d a t e i t h e r 2 0 0 0 0 o r 5 0 0 0 0 t i me s n o mi n a l

ma g n i f i c a ti o n o n A G F A O r t h o 2 5 f ilm a n d d e v e l o p e d f o r

17min a t 20C in I l fo rd Percep to l . Magn i f i ca t ion

ca l ib ra t ion was per fo rmed us ing a l i ne g r id .

Amino ac id ana lys i s

Sa mp l e s o f th e S - s u l p h o - p ro t e i n s w e r e h y d r o l y s e d w i t h

c o n s t a n t b o i l i n g 6 M H C I a t 1 0 8C u n d e r v a c u u m f o r 2 4 h

a n d a n a l y s e d o n a B i o t r o n i c L C 6 0 0 0 E a mi n o a c i d

ana lyser .

Prepara t ion o f rad io label led S -carboxymethy la ted

kera te ins

T h e r e d u c t i v e c l e a v a g e o f th e S - s u l ph o g r o u p s a n d t h e

fo l lowing S-carbox ym et hy la t ion s t ep ( iodo[2-14C]_ace t ic

a c i d ) w e r e c a r r i e d o u t u n d e r t h e s a me c o n d i t i o n s a s

descr ibed by M arsha l l and Gi l l esp ie 26.

Polyacrylamide gel electrophoresis

So l u t i o n s o f r a d io l a b e l le d p r o t e i n s w e r e e x a mi n e d b y

t w o - d i me n s i o n a l p o l y a c r y l a mi d e g e l e l e c t r o p h o r e s i s

accord ing to Ma rsha l l and Gi l l esp ie 26. P ro te in

separa t ion w as car r i ed ou t a t p H 8 .9 ( l s t d im. : 8 M urea ,

7 . 5 % s e p a r a t i o n g e l ; 2 n d d i m . : 0 . 1 % SD S, 1 0 %

separat ion gel) .

Af te r e l ec t rophores i s , rad io labe l l ed p ro te ins were

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

2 . 5 - d ip h e n y l o x a z o le a n d p la c e d o n a K o d a k - X - O m a t A R

film a t - 35C fo r 7 h a f t e r d ry ing the ge l a t 80C under

v a c u u m.

Pro te in prepara t ion for l aC-n .m.r s tud ies

Freeze-d r i ed S-su lpho-pro te in f rac t ions were d i s so lved

in the fol lowing solut ion (1 mg /ml) for 12-15 h at 6C: 8 ~o

f l -merca p toe thano l , 8 M urea , 0 .05 M Tr i s -H Cl , pH 7 .5 .

Af te r cen t r i fuga t ion (4000 rev /min , room temp era tu re ,

1 0 mi n ) t h e s u p e r n a t a n t w a s d i l u t e d w i t h 8 M u r e a t o

appro x im ate ly 1 mg/m l . P ro te ins w ere f reeze-d r ied a f te r

d i a ly s in g a g a i n s t a b u f fe r c o n ta i n i n g 0 .0 5 M N H 4 H CO 3 /

N H 3 O H , p H 8 .5 f o r 1 0 d a y s .

Re su l t s

Figure 1

s h o w s s c h e ma t i c a ll y t h e e x t r a c t io n o f w o o l

kera t ins us ing ox ida t ive su lph i to lys i s . Af t e r reduc t ive

c leavage o f the cys t ine b r idges wi th so d ium su lph i te ,

c y s t e i n e g r o u p s a r e c o mp l e t e l y c o n v e r t e d i n t o t h e S -

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

and sod ium su lph i t e . A separa t ion in to micro f ib r il l a r and

mat r ix p ro te ins can be ach ieved accord ing to the i r

d i f fe ren t so lub i l i t y behav iour us ing an i soe lec t r i c

p rec ip i t a t ion p rocedu re . Af te r rem oval o f the so lub le

ma t r i x c o mp o n e n t s t h e f i l a me n t o u s S - s u l p h o p r o t e i n s

can b e t rans fo rm ed in to the th io l fo rm us ing an excess o f a

reducing agen t .

W i t h t h e o x i d a t i v e s u lp h i to l y s is p r o c e d u r e m o r e t h a n

50% of the s t a r t ing mater i a l (L inco ln wool ) can b e

so lub i li zed . Scann ing e l ec t ron m icrosc opy sh ow s 25 tha t

P e l l e t : m i c r o f i b r i l l a r p r o t e i n s

( L S - S S O 3 )

R e d u c t i o n

s s s o s

R e c o n s t i t u t i o n o f I F

( L S - S S - L S )

W o o l

K e r a t i n

e x t r a c t i o n

o x i d a t i v e s u l p h i t o l y s i s )

k e r - S S - k e r ~ 2 k e r - S S 0 3~

I s o e l e c tr ic p r e c i p i t a t i o n

S u p e r n a t a n t : m a t r ix p r o t e in s

( H S - S S 0 3 0 )

Figure 1 W ool keratin extraction schem e. Protein extraction

procedure performing oxidative sulphitolysis followed by an

isoelectrical precipitation of the microfibrillar, low sulphur

proteins (LS-SSO~ ); matrix, high sulphur proteins (HS-SSO 3)

remain in solution. Cleavage of the S-sulpho groups can be

achieved by reaction with an excess of reducing agent. Low

sulphur proteins so prepared are the starting material for the

in

vitro

reconstitution experiments of wool IF

in t . j . Bio l . Macromol . , 1986 , Vol 8 , October

259


3/7

I n v i t r o

reconst i tu t ion of w ool in termediate . fi laments: H. Tho ma s

et a l .

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

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

w o o l f i b r e s i n m i n o r a m o u n t s .

T h e m a t e r i a l s s ol u b i li z e d w i t h s o d i u m s u l p h i t e / s o d i u m

t e t r a t h i o n a t e a r e c o m p o s e d o f m i c r o f i b r il l a r a n d m a t r i x

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

d i m e n s i o n a l p o l y a c r y l a m i d e g el e l e c tr o p h o r e s i s ( 2 D -

P A G E ) p r o t e i n p a t t e r n s w i t h th o s e o f M a r s h a l l f o r S -

c a r b o x y m e t h y l a t e d k e r a t e i n s Figure

2) 26.

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

p r e c i p i t a ti o n u s i n g zi n c a c e ta t e w a s p e r f o r m e d i n o r d e r t o

s e p a r a t e m a t r i x f r o m m i c r o f i b r i l l a r k e r a t e in s 2 7 ; 6 4 9/0 o f

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

i n d e p e n d e n t l y o f t h e e x t r a c t i o n r a t e . T o t e s t w h e t h e r t h i s

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

2 D - P A G E , w e r e c a r ri e d o u t . Table 1 s h o w s t h e a m i n o

a c i d c o m p o s i t i o n o f th e p r e c i p i ta t e a n d t h e s u p e r n a t a n t ;

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

a m i n o a c i d s , e sp e c i a l ly in t h e c y s t i n e c o n t e n t . T h e

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

a m i n o a c i d s ( A s p , G l u , L y s , L e u , A l a ) w h i l e t h e o t h e r

~ ~ / ~ i ~ / ~ ~ : / i ~ : ~ ~

F igure 2 2D -P A GE of a wool p ro te in ex trac t The s am ple i s

com posed of h igh su lphur (HS ) and low su lphur (LS ) p ro te ins .

Af te r reduc t ive c leavage of the p ro tec t ing S -su lpho groups

sam ples were rad io labe l l ed by S -ca rboxym ethy la t ion o f the

resulting thiol groups using iodo(2-14C)acetic acid.

Electrophores is according to Marshal l and Gil lespie26; charge

sepa ra t ion (u rea ) f i r s t d im ens ion , fo l lowed by S DS -P AGE a t

r ight angles

Table 1 Am ino ac id com p os i t ion (m ol 9 'o ) o f wool low/h igh

su lphur p ro te ins . The a r row s ind ica te an inc reased (1" ) o r

dec reased (~ ) concen t ra t ion in the low su lphur p ro te ins in

com par i son wi th the h igh su lphur f rac t ion

Low su lphur High su lphur

fract ion fract ion

As p 9.4 ~ 3.1

Th r + 5.5 11.3

Ser I 9.9 14.5

G lu 16.3 T 9.6

Pro ~ 4.9 14.5

Gl y 7.5 6.9

Ala 7.2 T 3.2

(Cys)2~ 2.9 6.3

Val 5.8 6.4

Met 0.6

Ile 3.2 3.6

Leu 10.4 ~ 4.2

Ty r 2.9 2.7

Phe 2.6 2.2

Ly s 1.9 i" 0.3

Hi s 0.8 0.8

Ar g 7.8 8.3

" A low cy stine content is detected because of the fact that S-sulpho-

cysteine is not completely detectable by amin o acid analysis

S I

L 8 H S

F igures 3 /4 2D -P A GE of m ic rof ib r i ll a r , low su lphur (LS ) and m a t r ix , h igh su lphur (HS ) p ro te ins . S am ple p repa ra t ion a s we ll a s

pe r form ance of the e lec t rophores i s , s ee Figure 2 . The e lec t rophore t i ca l expe r im ents c lea r ly dem ons t ra te the pos s ib i l i ty o f a

quan t i t a t ive s epa ra t ion in LS and HS pro te ins accord ing to the wool ex t rac t ion s chem e

Figure 1)

2 6 0 I n t . J . B i o l . M a c r o m o l . , 1 9 86 , V o l 8, O c t o b e r


4/7

I n v i t r o reconstitution of wool intermediate filaments: H Thomas: et a l .

C H

M i c r o f i b r i l l a r p r o t e i n s i

. / L e u

a h ~ A l a ~ /

I I L P ro . /

I

I I I I i

2 0 0 1 5 0 1 0 0 5 0 0

6 ( p p m )

M a t r i x p r o te i n s

ehl

\

C H

P h e / ~ ~ / / V ~ i a

I I I I i

1 2 0 0 1 5 0 1 0 0 5 0

0

6 ( p p m )

Figures 5/6 t3C-n.m.r, spectra of wool LS/H S prote ins. 75.5 M Hz ~aC-n.m.r . CP /MA S spectrum o f sol id fract ions of Lincoln wool .

Measu rements an d signal assignments were conducted accord ing to K richeldo rf and Mii llera4; for each spectrum abou t 12000

transients were accum ulated. P rote in samples w i th recom bined cyst ine bridges, received af ter reduct ive c leavage of the S-sulpho groups

followed by oxidat ive dia lysis, w ere examined. I n the sh ort range o rder microfibr i l lar prote ins show st rong ~-hel ical (~h) and weak f l-

st ructure (f ls) signals whereas in the case of the m atr ix prote ins the st rength of the signals i s

vice versa

Figure 7 Elect ron microg raph of protofi lamen tous st ructures of wool microfibri llar mater ia l . After reduct ive cleavage of the S-sulpho

groups and 100 000 g centr i fugat ion wool microfibr i l lar prote ins were dia lysed for 16 h di rect ly against a no n-urea co ntaining T ris-H Cl

buffer . Samples w ere negatively sta ined wi th 1% uranyl aceta te . Sho rt protofi lame ntous st ructures in a diam eter range of

approx imately 2-4 nm can be observed. Bar , 100 nm; x 140000

s o lu b l e c o m p o n e n t is m a i n l y c o m p o s e d o f am i n o a c id s

d i s t u r b i n g ~ t- he li ca l c o n f o r m a t i o n ( P r o , S e r , T h r ) .

Figures 3 a n d 4 s h o w t h e 2 D p a t t e r n s o f t h e s e p a r a t e d

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

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

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

m a t r i x c la s s , a s s h o w n b y c o m p a r i s o n o f th e s e r e s u l ts w i t h

t h o se a l r e a d y e s t a b l i sh e d i n t h e l i t e r a t u r e 2 6.

I n t . J . B i o l . M a c r o m o l . , 1 9 8 6, V o l 8 , O c t o b e r 2 6 1


5/7

In vitro

reconst i tu t ion o f wool in termediate f i lamen ts: H. Tho ma s

et al.

Figure

8 Effectof prolonged dialysis ime on filamentreconstitution of wool LS proteins. In contrast to the upper sample preparation

(16 h dialysis) a prolonged dialysis (24 h) was performed. Quite long filamentous structures can be observed with the filaments having

the tendency to twist around each other. Bar, 100 nm; x 105000

Figures 5

and 6 show ~3C-n.m.r. studies of the protein

fractions after cleavage of the protecting groups and

recombination of cystine linkages. Both keratin classes

show high degrees of short-range order. Besides 10-20%

of totally amorphous parts there is a relation of 8/2 (~-

helical//~-structure) in the microfibril lar proteins while the

matrix components show a relation of 3/7, indicating a

strongly diminished a-helical content.

The 13C-n.m.r. studies as well as the results of amino

acid analysis, 2D-PAGE and the constant precipitating

ratio of 64 ~o shows that a complete separation in the two

wool protein classes (low sulphur/high sulphur) can be

achieved by the use of the S-sulpho technique.

The low sulphur wool proteins thus isolated were used

as starting material for the

in vitro

reconstitution of wool

microfibrils.

Directly after the reductive cleavage of the protecting

groups, reconst itution of the intermediate filaments in t he

disulphide form was performed and the filamentous

proteins were negatively stained with uranyl acetate.

After dialysing the samples for 16 h against a non-urea-

containing Tris buffer, relatively short protofilamentous

structures could be observed with a diameter of 2 4 nm,

as shown in Figure 7 .

After a prolonged dialysis (total 24 h) under the same

conditions, filament assembly was more or less

completed, resulting in 7-11 nm IF, with the proteins

having the tendency to aggregate by twisting around each

other

Figure 8).

Obviously the conditions chosen were not yet optimal.

With the help of an additional dialysis step (against 4 M

urea) further improvement in IF reaggregation could be

achieved

Figure 9).

The filaments are no longer twisted;

they appear to be longer and the outlines are sharper.

D i s c u s s i o n

Keratin extraction procedures using oxidative

sulphitolysis have been performed to obtain pure wool

microfibrillar proteins as starting material for

in vitro

reconstitution experiments. Only about 50~ of the

starting material can be solubilized. This is less than with

commonly used reduction procedures 2s and could be

explained by the extraction step as such being very gentle,

resulting in single fibres still resistant against the chemical

attack, as revealed by scanning electron microscopy.

However, the described method fulfils the following

conditions in order to reconstitute wool-IF:

(1) representative isolation of protein material

throughout the fibre,

(2) prior condition for a complete separation into

microfibrillar and matrix proteins, and

(3) a cleavage of the S-sulpho groups can be easily

performed29.30.

262 Int. J. Biol. Macromol. , 1986, Vol 8, October


6/7

In vitro r e c o n s t i t u t i o n o f w o o l i n t e r m e d i a t e f i l a m e n t s : H . T h o m a s et al.

Figure 9 Electronmicrographs of intermediate-sizedfilamentsreconstituted from wool microfibrillar proteins. After cleavage of the

S-sulpho group and 100 000 g centrifugation filament reconstitution was achieved by two-step dialysis. First, samples were dialysed

against 4 M urea bufferand then dialysed against the filament buffer (I0 mM Tris-HC1, 10 mM fl-mercaptoethanol,pH 7.5). This two-

step procedure obviously led to the best results. Wool microfibrillar proteins can be reconstituted to long filaments showing the

diameter range of 7-11 nm being typical for intermediate filaments. Bar, 100 nm; x 78 000

The latter point represents the main difference in

comparison to the other keratin extraction and

separation procedures; e.g. a reduction followed by S-

carboxymethylation leads to an irreversible introduction

of charged groups into the polypeptide chains. In contrast

to these methods the keratin isolation procedure using

oxidative sulphitolysis allows the reformation o f cystine

bridges and and is therefore an approach to the 'native

structure'.

On the basis of this method it has been possible for the

first time to reconstitute IF from hard keratin fibres in

vi tro showing the typical diameter of 7-11 nm.

Obviously the filament formation is not spontaneous.

A prolonged dialysis time is therefore necessary, possibly

because the reconstitution of IF proceeds via a succession

of intermediate states involving thiol-disulphide

interchange.

With the successful IF reconstitution, hard ~-keratin

microfibrils can be classified as members of the IF family

not only on the basis of sequence data but also on their

electron microscopical appearance, this being the original

criterion.

Our results differ from those obta ined by Campbell e t

al.15 and Ahrnadi et al. 16 who investigated only ~-helical

enriched fragments of low-sulphur keratinous proteins.

However, according to the current state of knowledge, the

non-helical terminal domains of IF proteins play an

essential role in filament assembly and stabili ty 17'a1'32

As these are particulary rich in cysteine9,33 it is not

surprising that previous attempts on S-carboxy-

methylated wool keratin failed to provide in v i tro filament

formation.

Starting with the in v i tro reconstitution of wool IF,

further studies could elucidate the common properties of

the IF family of proteins. In the case of wool the chemical

and physical behaviour of isolated microfibrils can be

investigated by, e.g. using X-ray techniques to detect a

possible fibre diagram after orientation of IF proteins.

This would complement the 13C-n.m.r. data.

It must also be mentioned that an in vitro

reconstitution of wool IF cannot be achieved with the

same ease as in the case of other IF proteins, e.g. from

human skin. As we have performed IF reconstitution of

callus keratins even without separation of the keratins

from other cellular proteins (A. Conrads, unpublished

data) parallel to the wool microfibrillar proteins, we could

observe that the renatured wool IF often do not show the

more 'normal' appearance compared with the skin

Int. J. Biol. Macromol., 1986, Vol 8, October 263


7/7

I n v i t r o reconstitution o f wool intermediate filaments. H. Th om as et a l .

kerat in IF wh ich are very smo oth an d long. In order to

explain the observed phenomenon a further advance in

the kno wled ge o f the respect ive prote ins i s necessary.

A c k n o w l e d g e m e n t s

We are grate ful to Profes sor H. R . K riche ldorf for

carrying o ut the ~3C-n .m.r . s tudies and for perm iss ion to

publ i sh the data and to Dr J . F6h les for performing the

am i n o ac i d an a ly s is . F u r t h e r m or e w e w i s h to t h an k D r R .

C. Marshal l , Dr R . A. Quin lan and E. Bartn ik for

v a l u ab l e d i s c u s s i on s an d t h e D e p ar t m e n t o f P l an t

P h y s i o l og y a t t h e R W T H A ac h e n f or t h e p e r for m an c e o f

the 100000 g centr i fugat ion . In addi t ion , we thank

Profes sor Dr K. Weber , Drs E. F innimore and J . F /Sh les

for their critical reading of the man uscript.

R e f e r e n c e s

1 Ishikaw a, H., Bischoff, R. and Ho l tzer, R J Ce l l B i o l 1968, 38,

538

2 Lazaride s, E. A n n R e v B i o c h e m 1982, 51 ,219

3 Mo l l , R. , Fra nke , W. W. and Schi ller, D. L. C e l l 1982, 31, i1

4 Web er , K . and Gei s l e r, N . E M B O J 1982, 1, 1155

5 Gei s l e r , N . and Web er , K . E M B O J 1982, 1, 1649

6 Hanu koglu , I . and Fuchs , E . C e l l 1982, 31 ,243

7 Dow l ing , L . M. , Parry , D . A . D . and Sparrow, L . G . B i o s c i R e p

1983, 3, 73

8 Fraser , R . D . B . and Mac Rae , T . P . B i o s c i R e p 1983, 3, 517

9 Hanu koglu , I . and Fuchs , E . C e l l 1983, 33, 915

10 Crewther , W. G . , Dow l ing , L . M. , S t e iner t , P . M. and Parry , D .

A D I n t J B i o l M a c r o m o l 1983, 5, 267

11 Steinert , P. M., Rice, R. H., Roop , D. R., Trus , B. L. and Steven,

A C N a t u r e 1983, 302, 794

12 Zahn , H . M e l l i a n d T e x t i l b e r 1941, 22, 305

13 Jones , L . N . B i o c h e m B i o p h y s A c t a 1975, 412, 91

14 Steinert , P. M., Dye r, P. Y. and Rogers, G E J I n v e s t

D e r m a t o l 1971, 56, 49

15 Camp bel l , M. E. , Do bb, M. G . , H i lburn , M. E . , Loh , P ., Lot ay ,

S . S . , Speakman, P . T . , S t a insby , G . and Yarwood, R . E . i n

'Proceedings 5 th In t e rna t i ona l Wool Text i l e Research

Con ference Aa chen ' , (Ed. K. Ziegler), 1976, Vol . II , p. 243

16 Ahm adi , B . , Bos ton , N . M. , Dob b, M. G . and Speakman, P . T . i n

'Fibrous Proteins: Scient i fic, Indust rial and Medical Aspects ' ,

(Eds D . A . D . Parry and L . K . Creamer) , Academic Press ,

Lon don , 1980, p. 161

17 S t e iner t, P . M. i n 'E l ec t ron Microscopy of Pro t e ins ' , (Ed . J . R .

Harris), Academic Press, London, 1981, p. 126

18 God dard , D . R . and Michae l i s , L J B i o l C h e m 1935, 11 2, 361

19 Alexander , P . and Ear l and , C . N a t u r e 1950, 166, 396

20 Bailey, J. L. B i o c h e m J 1957, 67, 21

21 Harrap , B . S . and Wo ods , E . F . B i o c h e m J 1964, 92, 8

22 Spei, M. and Thom as , H . C o l l o i d a n d P o l y m S c i 1983, 261,96 8

23 Spei, M. and Thom as , H . M e l l i a n d T e x t i l b e r 1984, 65, 266

24 Thom as , H . PhD Thesi s, RW TH Aachen , 1984

25 Thom as , H . , Greven , R . and Spei, M. M e l l i a n d T e x t i l b e r 1983,

64, 297

26 Mars hal l , R. C. and Gi l lespie, R M J F o r e n s S c i S o c 1982, 22,

377

27 Gillespie, J. M. A u s t J B i o l S c i 1957, 10, 105

28 Greven , R . PhD Thesi s, RW TH , Aachen , 1982

29 Fo otne r, H. B. and Smiles, S J J C h e m S o c 1925, 127, 2887

30 Thom as , H . and Spei, M. M e l l i a n d T e x t i l b e r 1984, 65, 208

31 Sauk , J . J . , Krum mweide , M. , Cocking -Johnson , D . and Whi t e ,

J G J Ce l l B i o l 1984, 99, 1590

32 Gei s l e r, N . , Kaufm ann, E . and Weber , K . C e l l 1982, 30, 277

33 Fraser , R . D . B ., MacR ae , T . P . and Rogers , G . E . i n 'Kera t i ns ,

t he i r Composi t i on , S t ruc ture and Biosynthes is ' , C . C . Thom as ,

Springfield, Il l inois, 1972

34 Kri che ldorf , H . R . and Muel l e r , D . M a c r o m o l e c u l e s 1983, 16, 615

2 6 4 I n t . J . B i o l . M a c r o m o l . , 1 9 8 6, V o l 8 , O c t o b e r

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