V o l u m e 294, n u m b e r 3, 2 7 9 - 2 8 1 F E B S 10493 ~5 1991 Federation o f E u r o p e a n Biochemical Societies 00145793/91/$3.50

D e c e m b e r 1991

M a t r i x d e g r a d i n g p r o p e r t i e s o f s p e r m serine proteina3e, acrosin T . P l a n c h e n a u l t I, D . ( ~ e c h o v h 2 a n d V. K e i l - D l o u h a ~ ~Laboratoire d e C h i m i e d e s P r o t d i n e s . l n s t i t . ' t P a s t e u r , 28 rue d u D o c t e u r R o u x . 7 5 7 2 4 P a r i s C e d e x 15. F r a n c e a n d 2Institute o f M o l e c u l a r Genetics. C z e c h o s t yak A c a d . SoL. F l e m i n g o v o n d m . 2. / 6 6 3 7 P r a g u e 6. C z e c h o s l o v a k i a R e c e i v e d 14 O c t o b e r 1991 T h e serine proteinase acrosin plays an i m p o r t a n t role in sperm penetration o f the zona pellucida. In the present study we investigated the effect o f the e n z y m e on various matrix proteins. Acrosin d e g r a d e d proteolytically fibronectin. ,ype IV collagen and heat denatured type I collagen, whereas neither native type I collagen n o r laminin were cleaved by the enzyme. The specific activily o f acrosin with type IV collagen as substrate (66.6 g/h/g} was 125-fold higher than that o f k n o w n type IV eollagenase o r stromelysin. These results suggest that acrosin may act as a matrix-degrading proteinase. A c r o s i m Matrix-degrading activity; Collagen IV; Fibronectin

1. I N T R O D U C T I O N Acrosin plays a central role in mammalian fertilization. The enzyme was found in spermatozoa of verteb r a t e s a n d n o n - v e r t e b r a t e s ( r e v i e w e d in [ 1]). It is a disul-

f i d e - b o n c d t w o - c h a i n g l y c o s y l a t e d serinc p r o t e i n a s e , exh i b i t i n g t r y p s i n - l i k e c l e a v a g e specificity [2,3]. In intact s p e r m a t o z o a , t h e e n z y m e is a s s o c i a t e d w i t h t h e a e r o s o real p e r i p h e r a l m e m b r a n e in f o r m o f p r o a c r o s i n w h i c h is activated

by

limited

proteolysis

before

fertilization

[4,5t. The high amount of acrosin in the acrosome of spermatozoa is believed to be essential for g~rnete fusion, particularly for binding to and penetration of the zona p e l l u c i d a ([6], r e v i e w e d i n [7]). T h e m o l e c u l e o f a c r o s i n combines a specific proteolytic activity with zo•a- and carbohydrate-affinity, a property unusual for a serine p r o t e i n a s ¢ [8,9]. It w a s s u g g e s t e d t h a t in v i v o t h i s s p e c i a l affinity of acrosin directs the proteolytic activity to its structural target. Sperm acrosin may thus function in penetration of zona pellucida and of a highly structured e x t r a c e l l u l a r m a t r i x o f t h e p r e v i t e l l i n s p a c e [6]. Since little is known about the chemical structure of the zonapdlucida, the action of acrosin on its presumed physiological substrate has not been characterized on the molecular level and the degradation of matrix proteins was attributed rather to the action of matrix-degrading metalloproteinases. We have recently described that matrix protein, fibro-

nectin, contains a latent proteolytic system o f matrix d e g r a d a t i o n , t h a t c a n be a c t i v a t e d b y e x t e r n a l p r o t e i n a s e s [ 1 0 , 1 !]. F N - t y p e

IV eoUagenase,

one enzyme

of this

Correspondence address: V. Keit-Dlouha, L a b o r a t o i r e de Chimie des Prot~ines, Institut Pasteur 28 rue du D o c t e u r R o u x , 75724 Paris Cedex 15, France. Fax: (33) ( i ) 405 60125.

Publi3hed by Elsevier Science Publishers B.V.

s y s t e m , is a m e t a l l o p r o t e i n a s e that degrades collagen type IV, heat-denatured collagen tyt'~ ! and laminin. Fibronectinase i s a s e r i n e p r o t e i n a s ¢ t h a t d e g r a d e s fibronectin and its fragments. As both the sperrnatozoid and the previtellin space contain fibronectin, a process of a potential activation of its latent proteolytic system by acrosin had to be taken into consideration. The intent of this study was to examine the direct effect of the serine proteinase acrosin on various matrix proteins and the possibility of an indirect effect of activation of a matrix-degrading activity from fibronectin

[10,11]. 2.

MATERIALS

AND

METHODS

Gelatin-Ultrogel was purchased from IBF Biotechnics (Villeneuvcla-Garean©, Franc.c). Phenylmethane sulfonylfluoride ( P M S F ) and pepstatin A were from F l u k a C h e m i c A G (Buchs, Switzerland), I, ! 0p h e n a n t h r o l i n e a n d n-ethylmaleimide were from Sigma Chemical Co. (St. Louis. USA). Acrosin (at-form, 14 U/rag) was purified and activated as previously described [12]. T h e lyophilized sample was solubilized in I m M HCI t o a final cone. o f I mg/mi. Lyophilized h u m a n plasma fibronectin (Centre National de Transfusion Sanguine, France), was purified as described [ 13,14]. with a final step o f purification by gel filtration [ ! 5]. Laminin a n d type IV collagen were purified from the EngelbrethH o l m - S w a r m ( E H S ) t u m o r [16] a n d type ! collagen from rat t e n d o n s [ 17]. H e a t - d e n a t u r e d collagen (gelatin) was prepared by incubation o f native type ! collagen at 70°C for 15 rain. E n z y m e assays with native type ! collagen, denatured type ! collagen [gelatin}, type IV collagen a n d laminin as substrates (enzyme/substrate ratio o f 1:!000) were carried out in 50 m M Tris-HCl, p H 7.4, buffer, c o n t a i n i n g 0.2 M N a C i and 5 m M CaCI2 with 20 g o f substrate in a final volume o f 50 litres. In the case o f laminin, the assays were p e r f o r m e d w i t h o u t NaCI. T h e samples were incubated for 16 h a t 37°C, lyophilized, reduced and analyzed by electrophoresis using 10~ acr3damide gel according to Doucet a n d Trifaro [18]. Substratc digeslion was ©valuated in Laser I ~ n s i t o m e t g r Ultroscan X L ( L K B , Sweden) by c o m p a r i s o n o f control and enzyme-containing samples. The inhibition studies were d o n e as previously described [10,1 I]

279

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with phenylmethane sulfonyifluoride. 1,10-phenanthroline, pepstatin A a n d h i - e t h y l r n a l e i m i d e i n f i n a l c o n c e n t r a t i o n s o f t .3 m M . 1 0 r n M , I mM and 3.3 mM, respectively. To evaluate the partial digestion of fibronectin with acrosin, the p r o t e i n (5 r a g ) w a s d i g e s t e d a t e n z y m c / s u b s t r a t e r a t i o o f I : I 0 0 i n 5 rnl o f 5 0 m M T r i s - H C I b u f f e r , p H 8, c o n t a i r ~ i n g 5 m l ~ C a C I 2 a t 2 2 ° C . A f t e r 2 h , t h e d i g e s t w a s a p p l i e d o n a g e l a t i n - U P . v o g e l c o l u m n (2~2 × I 0 c r n ) , ecluii i b r a t e d w i t h t h e s a m e b u f f e r . T h e r e t a i n e d f r a g m e n t s w e r e eluted with the equilibration buffer containing 4 M urea.

3. R E S U L T S

AND

•

2

3

4

5

6

Gg

DISCUSSION

The effect of acrosin on collagens was studied with native and heat-denatured collagens type 1 and with native collagen type IV as substrates. -The results in Fig. IA show that native type I collagen was not cleaved by the enzyme. On the contrary, the heat-denatured collagen was degraded. The degradation of basal membrane matrix proteins was studied with collagen type IV and with laminin, the s u b s t r a t e s o f a specific g r o u p o f m a t r i x - d e g r a d i n g metalloproteinases, comprising type IV collagenases and stromelysin. O u r r e s u l t s ( F i g . I B ) h a v e s h o w n t h a t a c r o s i n effic i e n t l y d e g r a d e s t y p e I V c o l l a g e n a t E / S r a t i o o f 1 : 1000. This degradation, as well as the cleavage of the heat° denatured type 1 collagen, were inhibited by the serine pr,~teinase inhibitor phenylmethane sulfonylfluoride, while the inhibitor of metalloproteinases, o-phenant h r o l i n e , w a s w i t h o u t effect. T h e r e f o r e the c l e a v a g e o f type IV and denatured type I collagens can be attributed to acrosin itself and not to an eventual contamination by a metalloproteinase or by its zymogen. Contrary to collagen type IV, laminin was not degraded by acrosin (Fig. IC). T h e e f f e c t o f a c r o s i n o n f i b r o n e c t i n is d e m o n s t r a t e d i n F i g . 2. T h e p r o t e o l y s i s o f f i b r o n e c t i n a t a n E / S r a t i o o f 1 : 100 w a s a c c o m p l i s h e d w i t h i n 2 h ( F i g . 2 A ) . F u r t h e r incubation did not change the electrophoretic pattern of the digest (not shown). An affinity chromatography of the digest on gelatinU l t r o g e l ( F i g . 2 B ) a n d a s u b s e q u e n t gel e l e c t r o p h o r e s i s of the separated fractions (Fig. 2A) have demonstrated t h r e e m a i n f r a g m e n t s o f 110, 2 7 a n d 2 5 k D a i n g e l a t i n unretained fraction. All other fragments were retained b y g e l a t i n - U l t r o g e l . XVhen e i t h e r t h e g e l a t i n - u n r e t a i n e d or gelatin-retained fractions were assayed with various matrix proteins as substrates, no traces of any newly generated proteolytic activity were detected. A 24 hpreincubation of the fractions alone or together (ratio 1:i) that enhanced the activation of FN-proteinases did not change the results. Therefore the present study has demonstrated that acrosin does not generate any proteolytic activity from f i b r o n e c t i n , b u t t h a t it c a n d e g r a d e d i r e c t l y m a t r i x p r o teins. A c o m p a r i s o n o f t h e specific activities o f a c r o s i n towards the studied matrix proteins with those of k n o w n m a t r i x - d e g r a d i n g p r o t e i n a s e s ( T a b l e I) d e m o n 280

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Fig. I. Analysis of proteolytie activity of acrosin. Substrates were incubated with acrosin (E/S ratio of 1:100) for 16 h at 37°C. (A) Native type I collagen (20 g) alone (lane 1) or with acrosin (lane 2); (lane~ 3-6) heat denatured collagen (20 g) as substrate: incubated atone (lane 3). with aerosin (lane 4), with acrosin and phenylmethane snlfonyltluoride (lane 5), with acrosin and 1,10-phenanthroline (lane 6). (B) Collagen type IV (! 5 g) alone (lane 1); with the enzyme (lane 2); with the enzyme and PMSF (lane 3); with the enzyme and I ,lO-phenanthroline 0ane 4). (C) Laminin (15 g) alone (lane l); laminin with the enzyme (lane 2). s t r ~ t e s t h a t its s p e c i f i c a c t i v i t y t o w a r d s t y p e I V c o l l a g e n is 1 2 5 - f o l d h i g h e r t h a n t h a t o f t y p e I V c o l l a g e n a s e ( g e l a t i n a s e ) a n d o f s t r o m e l y s i n [19]. Up to now, the degradation of type IV collagen was considered as a specific property of metalloproteinases, in particular of type IV collagenases. This group of enzymes was exhaustively studied by several laboratories, s i n c e a c o r r e l a t i o n w a s e s t a b l i s h e d b e t w e e n t h e i r proteolytic activity and the metastatic potential of t u m o r c e l l s [20,21]. Our present results brought evidence that serine proteinase acrosin can also display a hish specific proteolytie activity towards collagen type IV. Therefore a degradation of this matrix protein characteristic for the b a s a l m e m b r a n e is n o t a n e x c l u s i v e p r o p e r t y o f m e t a l l o p r o t e i n a s e s . F u r t h e r s t u d y is r e q u i r e d t o u n d e r s t a n d t h e biological significance of this particular property of acrosin.

Volume

294, number

3

FEBS

LETTERS

A

December

1991

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Fig. 2. D i g e s t i o n o f f i b r o n e c t i n w i t h a c r o s i n a n d affinity c h r o m a t o g r a p h y o f t h e d i g e s t o n 8 ¢ l a t i n - U l t r o g ¢ l . ( A ) G e l e l e c t r o p h o r c s i s . Fil~roncctin ( l a n e I); a c r o s i n d i g e s t o f f i b r o n e c t i n ( l a n e 2); 8 e l a t i n - u r t r e t a i n e d f r a c t i o n