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New method for obtaining IgA-specific protease
Journal oflmmunological Methods, 18 (1977) 245--249 O Elsevier/North-Holland Biomedical Press
245
NEW M E T H O D F O R O B T A I N I N G I g A - S P E C I F I C P R O T E A S E *
T.B. HIGERD, G. VIRELLA, R. CARDENAS, J. KOISTINEN ** and J.W. FETT Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29401, U.S.A. (Received 14 March 1977, accepted 14 June 1977)
A simple method for obtaining an active preparation of IgA-specific protease from a bacterial source is presented. In this method Streptococcus sanguis was inoculated onto the surface of a dialysis membrane on nutrient agar. Following growth, the membrane was removed from the agar surface and washed in a small volume of buffer. A solution with protease activity against IgA1 monoclonal proteins was obtained by clarification of the wash and appeared to be similar to enzyme preparations obtained by other methods.
INTRODUCTION A specific h y d r o l a s e f o r i m m u n o g l o b u l i n A (IgA), f o u n d initially in h u m a n feces ( M e h t a e t al., 1 9 7 3 ) , r e p r e s e n t s an invaluable t o o l f o r t h e structural s t u d y o f I g A p r o t e i n s . This e n z y m e has b e c o m e increasingly i m p o r t a n t , since a l t e r n a t i v e m e a n s f o r e n z y m a t i c a l l y splitting IgA p r o t e i n s o f t e n fail to p r o d u c e a d e q u a t e a m o u n t s o f Fc-like f r a g m e n t s (Bernier et al., 1 9 6 5 ; Ballieux et al., 1 9 6 8 ; S h u s t e r , 1971}. Several b a c t e r i a l species are c a p a b l e o f p r o d u c i n g this e n z y m e : S t r e p t o c o c c u s sanguis (Plaut e t al., 1 9 7 4 ) , Neisseria g o n o r r h o e a e a n d N. m e n i n g i t i d i s (Plaut et al., 1 9 7 5 ) , a n d P s e u d o m o n a s aeruginosa ( L u b e c , 1976). In e a c h case, t h e m e t h o d f o r o b t a i n i n g an active p r e p a r a t i o n involves c o n c e n t r a t i o n t h r o u g h s a l t i n g - o u t o f p r o t e i n s f r o m s p e n t g r o w t h m e d i a b y a m m o n i u m sulf a t e a c c o r d i n g t o t h e p r o c e d u r e o f P l a n t e t al. ( 1 9 7 4 ) , resulting in a c r u d e p r e p a r a t i o n w i t h l i m i t e d yield a n d q u e s t i o n a b l e p u r i t y . T h e p r e s e n t repo~'t describes a m e t h o d t h a t d o e s n o t r e q u i r e large v o l u m e s o f c u l t u r e m e d i u m a n d yields highly active p r e p a r a t i o n s t h a t can be u s e d t o h y d r o l y z e I g A w i t h o u t c o n c e n t r a t i o n or p u r i f i c a t i o n .
* Publication no. 131 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. ** Present address: Finnish Red Cross Blood Transfusion Service, Kivihaantie 7, SF00310 Helsinki 31, Finland.
24'6 MATERIALS AND METHODS
Organism and medium S. sanguis was obtained from the American Type Culture Collection (ATCC 10556) and cultivated on tryptone--glucose--agar medium containing 20 g Bacto-tryptone (Difco), 5 g glucose, 4 g K2HPO4, 1 g KHzPO4, 2 g NaCl, 250 mg MgSO4 • 7H20, 17 mg MnSO4 and 15 g Bacto-agar (Difco) in 1 liter of distilled water. Preparation of IgA-specific protease S. sanguis was streaked for confluent growth on tryptone--glucose--agar and incubated anaerobically at 37°C. After 24 h, the cells were harvested in 10 ml of 0.05 M potassium phosphate buffer, pH 7.5, and adjusted to approximately 1 × l 0 s cells/ml. Discs of sterile dialysis membranes (Markson) having the same diameter as the Petri dish (100 mm) were laid on the agar surface of fresh plates, and 0.1 ml of the cell suspension was spread over the dialysis membrane with a c o t t o n swab. The plates were incubated at 37°C for 18 h under anaerobic conditions. After incubation, the dialysis membranes were removed and washed with a minimum volume of the same buffer. The wash was clarified by centrifugation at 30,000g for 15 min at 4°C, and the supernatant was designated as 'crude IgA protease preparation'. The use of single plates was adequate for screening potential bacterial IgA protease producers. However, approximately 100 plates were used in preparing the standard IgA protease solution employed in subsequent studies. The clarified wash was concentrated 10-fold by positive pressure ultrafiltration using an Amicon PM-10 filter which resulted in a non-pigmented solution with an A2s0 n m of 3.7. IgA proteins IgA proteins were isolated from sera of multiple m y e l o m a patients using a m m o n i u m sulfate for salting-out followed by precipitation with caprylic acid and ion-exchange chromatography on DEAE-cellulose columns {Fine and Steinbuch, 1970). The proteins were t y p e d for subclass and allotypes using serological reagents of known specificity by positive haemagglutination inhibition using the chromium chloride m e t h o d for coating the indicator red cells {Gold and Fudenberg, 1967).
Digestion of IgA proteins Equal volumes of a given IgA protein dissolved in phosphate-buffered saline at a concentration of 5 mg/ml and the crude IgA protease preparation were mixed and incubated at 37°C for 18 to 24 h. Digestion was stopped by
247 addition of 5 mM EDTA and the digested proteins were stored a t - - 2 0 ° C until used. The results of digestion were characterized by (a) immunoelectrophoresis (Grabar and Williams, 1953) using unabsorbed rabbit a~ti-IgA serum (prepared in this lab) and specific anti-~-chain serum (Wellcome), and by (b) sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis {Summers et al., 1965; Virella et al., 1975). RESULTS AND DISCUSSION The enzymatic capability of the preparation obtained by the m e t h o d described herein appears to be equivalent to that of preparations obtained by the relatively more complex procedure of Plaut et al. (1974). The use of the semi-permeable membrane between the micro-organism and the nutrient agar permits the exchange of nutrients necessary for cell growth and permits the separation of bacterial products of high molecular weight from the large proteins comprising nutrient media. In our laboratory, preparations of IgA protease prepared by the m e t h o d of Plaut et al. (1974) resulted in a pigmented solution following DEAE-cellulose column chromatography, whereas preparations made by our m e t h o d were colorless w i t h o u t purification. In
C 4-
D
Fig. 1. Immunoelectrophoretic analysis of the digestion products following incubation of purified monoclonal IgA1 with and without crude IgA protease preparation. Wells and troughs contain: C, IgA alone; D, IgA digested with IgA protease; ~, monospecific anti-schain; A, unabsorbed rabbit anti-IgA.
248
113
114
115
117
109
M
-IFig. 2. SDS polyacrylamide gel electrophoretic study of the effects of IgA protease on several IgA proteins of different subclasses and allotypes. The proteins included 113 = IgA2, A~m(2); 114 = IgA1K; 115 = IgAl~; 117 = IgAl~; 109 = IgA2, A2m(1)K. The proteins were incubated with (D) or without (C) the IgA protease prior to electrophoresis. Samples of IgA protease at the concentration used did not result in visible protein bands. The mobility of monomeric IgA is indicated (M).
a d d i t i o n , t h i n - l a y e r p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s revealed approxim a t e l y 20 p r o t e i n bands in the c r u d e dialysis p r e p a r a t i o n b u t was u n a b l e to resolve individual p r o t e i n s in the p r e p a r a t i o n o b t a i n e d b y the m e t h o d o f Plaut et al. (1974). Fig. 1 depicts an i m m u n o e l e c t r o p h o r e t i c profile o f the digestion p r o d u c t s following i n c u b a t i o n o f purified IgA w i t h the c r u d e IgA protease preparat i o n , indicating t h a t IgA can be e n z y m a t i c a l l y cleaved into Fab- and Fc-like f r a g m e n t s b y the p r o t e a s e p r e p a r a t i o n o b t a i n e d with this m e t h o d . When a large n u m b e r o f samples were digested, S D S - p o l y a c r y l a m i d e gel electrophoresis was f o u n d t o be a d e q u a t e t o d e t e r m i n e w h e t h e r or n o t digestion had o c c u r r e d . Fig. 2 shows the results o f the e l e c t r o p h o r e s i s w h e n the digest i o n p r o d u c t s o f several IgA p r o t e i n s o f d i f f e r e n t subclasses and allotypes were screened. O n l y IgA1 p r o t e i n s s h o w e d evidence o f hydrolysis; IgA2 a p p e a r e d t o be resistant t o IgA p r o t e a s e digestion. These results are in agreem e n t with t h o s e r e p o r t e d b y Plaut et al. (1974).
249 ACKNOWLEDGMENTS This w o r k was s u p p o r t e d in p a r t b y t h e S o u t h C a r o l i n a A p p r o p r i a t i o n f o r B i o m e d i c a l R e s e a r c h , N I H B i o m e d i c a l R e s e a r c h S u p p o r t G r a n t t o the College o f D e n t a l Medicine a n d U S P H S G r a n t s A I - 1 3 4 8 4 a n d H D - 0 9 9 3 8 . We are grateful to Dr. H. H u g h F u d e n b e r g f o r h e l p f u l discussions and t o Charles L. S m i t h f o r critical review o f t h e m a n u s c r i p t . REFERENCES Ballieux, R.E., J.W. Stoop and B.J.M. Zeegers, 1968, Scand. J. Hematol. 5,179. Bernier, G.M., V. Tominaga, C.W. Easley and F.W. Putnam, 1965, Biochemistry 9, 2072. Fine, J.M. and M. Steinbuch, 1970, Rev. Eur. Etud. Clin. Biol. 15, 1115. Gold, E.R. and H.H. Fudenberg, 1967, J. Immunol. 99,859. Grabar, P. and C.A. Williams, 1953, Biochim. Biophys. Acta 10,193. Lubec, G., 1976, Pathology 4,526. Mehta, S.K., A.G. Plaut, N.J. Calvanico and T.B. Tomasi, 1973, J. Immunol. 111, 1274. Plaut, A.G., R.J. Genco and T.B. Tomasi, 1974, J. Immunol. 113,289. Plaut, A.G., J.V. Gilbert, M.J. Artenstein and J.D. Capra, 1975, Science 190, 1103. Shuster, J., 1971, Immunochemistry 8,405. Summers, D.T., J.V. Maizel and J.E. Darrell, 1965, Proc. Nat. Acad. Sci. U.S.A. 59,505. Virella, G., R. Valadas-Preto and F. Graca, 1975, Brit. J. Haematol. 30,479.
245
NEW M E T H O D F O R O B T A I N I N G I g A - S P E C I F I C P R O T E A S E *
T.B. HIGERD, G. VIRELLA, R. CARDENAS, J. KOISTINEN ** and J.W. FETT Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29401, U.S.A. (Received 14 March 1977, accepted 14 June 1977)
A simple method for obtaining an active preparation of IgA-specific protease from a bacterial source is presented. In this method Streptococcus sanguis was inoculated onto the surface of a dialysis membrane on nutrient agar. Following growth, the membrane was removed from the agar surface and washed in a small volume of buffer. A solution with protease activity against IgA1 monoclonal proteins was obtained by clarification of the wash and appeared to be similar to enzyme preparations obtained by other methods.
INTRODUCTION A specific h y d r o l a s e f o r i m m u n o g l o b u l i n A (IgA), f o u n d initially in h u m a n feces ( M e h t a e t al., 1 9 7 3 ) , r e p r e s e n t s an invaluable t o o l f o r t h e structural s t u d y o f I g A p r o t e i n s . This e n z y m e has b e c o m e increasingly i m p o r t a n t , since a l t e r n a t i v e m e a n s f o r e n z y m a t i c a l l y splitting IgA p r o t e i n s o f t e n fail to p r o d u c e a d e q u a t e a m o u n t s o f Fc-like f r a g m e n t s (Bernier et al., 1 9 6 5 ; Ballieux et al., 1 9 6 8 ; S h u s t e r , 1971}. Several b a c t e r i a l species are c a p a b l e o f p r o d u c i n g this e n z y m e : S t r e p t o c o c c u s sanguis (Plaut e t al., 1 9 7 4 ) , Neisseria g o n o r r h o e a e a n d N. m e n i n g i t i d i s (Plaut et al., 1 9 7 5 ) , a n d P s e u d o m o n a s aeruginosa ( L u b e c , 1976). In e a c h case, t h e m e t h o d f o r o b t a i n i n g an active p r e p a r a t i o n involves c o n c e n t r a t i o n t h r o u g h s a l t i n g - o u t o f p r o t e i n s f r o m s p e n t g r o w t h m e d i a b y a m m o n i u m sulf a t e a c c o r d i n g t o t h e p r o c e d u r e o f P l a n t e t al. ( 1 9 7 4 ) , resulting in a c r u d e p r e p a r a t i o n w i t h l i m i t e d yield a n d q u e s t i o n a b l e p u r i t y . T h e p r e s e n t repo~'t describes a m e t h o d t h a t d o e s n o t r e q u i r e large v o l u m e s o f c u l t u r e m e d i u m a n d yields highly active p r e p a r a t i o n s t h a t can be u s e d t o h y d r o l y z e I g A w i t h o u t c o n c e n t r a t i o n or p u r i f i c a t i o n .
* Publication no. 131 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. ** Present address: Finnish Red Cross Blood Transfusion Service, Kivihaantie 7, SF00310 Helsinki 31, Finland.
24'6 MATERIALS AND METHODS
Organism and medium S. sanguis was obtained from the American Type Culture Collection (ATCC 10556) and cultivated on tryptone--glucose--agar medium containing 20 g Bacto-tryptone (Difco), 5 g glucose, 4 g K2HPO4, 1 g KHzPO4, 2 g NaCl, 250 mg MgSO4 • 7H20, 17 mg MnSO4 and 15 g Bacto-agar (Difco) in 1 liter of distilled water. Preparation of IgA-specific protease S. sanguis was streaked for confluent growth on tryptone--glucose--agar and incubated anaerobically at 37°C. After 24 h, the cells were harvested in 10 ml of 0.05 M potassium phosphate buffer, pH 7.5, and adjusted to approximately 1 × l 0 s cells/ml. Discs of sterile dialysis membranes (Markson) having the same diameter as the Petri dish (100 mm) were laid on the agar surface of fresh plates, and 0.1 ml of the cell suspension was spread over the dialysis membrane with a c o t t o n swab. The plates were incubated at 37°C for 18 h under anaerobic conditions. After incubation, the dialysis membranes were removed and washed with a minimum volume of the same buffer. The wash was clarified by centrifugation at 30,000g for 15 min at 4°C, and the supernatant was designated as 'crude IgA protease preparation'. The use of single plates was adequate for screening potential bacterial IgA protease producers. However, approximately 100 plates were used in preparing the standard IgA protease solution employed in subsequent studies. The clarified wash was concentrated 10-fold by positive pressure ultrafiltration using an Amicon PM-10 filter which resulted in a non-pigmented solution with an A2s0 n m of 3.7. IgA proteins IgA proteins were isolated from sera of multiple m y e l o m a patients using a m m o n i u m sulfate for salting-out followed by precipitation with caprylic acid and ion-exchange chromatography on DEAE-cellulose columns {Fine and Steinbuch, 1970). The proteins were t y p e d for subclass and allotypes using serological reagents of known specificity by positive haemagglutination inhibition using the chromium chloride m e t h o d for coating the indicator red cells {Gold and Fudenberg, 1967).
Digestion of IgA proteins Equal volumes of a given IgA protein dissolved in phosphate-buffered saline at a concentration of 5 mg/ml and the crude IgA protease preparation were mixed and incubated at 37°C for 18 to 24 h. Digestion was stopped by
247 addition of 5 mM EDTA and the digested proteins were stored a t - - 2 0 ° C until used. The results of digestion were characterized by (a) immunoelectrophoresis (Grabar and Williams, 1953) using unabsorbed rabbit a~ti-IgA serum (prepared in this lab) and specific anti-~-chain serum (Wellcome), and by (b) sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis {Summers et al., 1965; Virella et al., 1975). RESULTS AND DISCUSSION The enzymatic capability of the preparation obtained by the m e t h o d described herein appears to be equivalent to that of preparations obtained by the relatively more complex procedure of Plaut et al. (1974). The use of the semi-permeable membrane between the micro-organism and the nutrient agar permits the exchange of nutrients necessary for cell growth and permits the separation of bacterial products of high molecular weight from the large proteins comprising nutrient media. In our laboratory, preparations of IgA protease prepared by the m e t h o d of Plaut et al. (1974) resulted in a pigmented solution following DEAE-cellulose column chromatography, whereas preparations made by our m e t h o d were colorless w i t h o u t purification. In
C 4-
D
Fig. 1. Immunoelectrophoretic analysis of the digestion products following incubation of purified monoclonal IgA1 with and without crude IgA protease preparation. Wells and troughs contain: C, IgA alone; D, IgA digested with IgA protease; ~, monospecific anti-schain; A, unabsorbed rabbit anti-IgA.
248
113
114
115
117
109
M
-IFig. 2. SDS polyacrylamide gel electrophoretic study of the effects of IgA protease on several IgA proteins of different subclasses and allotypes. The proteins included 113 = IgA2, A~m(2); 114 = IgA1K; 115 = IgAl~; 117 = IgAl~; 109 = IgA2, A2m(1)K. The proteins were incubated with (D) or without (C) the IgA protease prior to electrophoresis. Samples of IgA protease at the concentration used did not result in visible protein bands. The mobility of monomeric IgA is indicated (M).
a d d i t i o n , t h i n - l a y e r p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s revealed approxim a t e l y 20 p r o t e i n bands in the c r u d e dialysis p r e p a r a t i o n b u t was u n a b l e to resolve individual p r o t e i n s in the p r e p a r a t i o n o b t a i n e d b y the m e t h o d o f Plaut et al. (1974). Fig. 1 depicts an i m m u n o e l e c t r o p h o r e t i c profile o f the digestion p r o d u c t s following i n c u b a t i o n o f purified IgA w i t h the c r u d e IgA protease preparat i o n , indicating t h a t IgA can be e n z y m a t i c a l l y cleaved into Fab- and Fc-like f r a g m e n t s b y the p r o t e a s e p r e p a r a t i o n o b t a i n e d with this m e t h o d . When a large n u m b e r o f samples were digested, S D S - p o l y a c r y l a m i d e gel electrophoresis was f o u n d t o be a d e q u a t e t o d e t e r m i n e w h e t h e r or n o t digestion had o c c u r r e d . Fig. 2 shows the results o f the e l e c t r o p h o r e s i s w h e n the digest i o n p r o d u c t s o f several IgA p r o t e i n s o f d i f f e r e n t subclasses and allotypes were screened. O n l y IgA1 p r o t e i n s s h o w e d evidence o f hydrolysis; IgA2 a p p e a r e d t o be resistant t o IgA p r o t e a s e digestion. These results are in agreem e n t with t h o s e r e p o r t e d b y Plaut et al. (1974).
249 ACKNOWLEDGMENTS This w o r k was s u p p o r t e d in p a r t b y t h e S o u t h C a r o l i n a A p p r o p r i a t i o n f o r B i o m e d i c a l R e s e a r c h , N I H B i o m e d i c a l R e s e a r c h S u p p o r t G r a n t t o the College o f D e n t a l Medicine a n d U S P H S G r a n t s A I - 1 3 4 8 4 a n d H D - 0 9 9 3 8 . We are grateful to Dr. H. H u g h F u d e n b e r g f o r h e l p f u l discussions and t o Charles L. S m i t h f o r critical review o f t h e m a n u s c r i p t . REFERENCES Ballieux, R.E., J.W. Stoop and B.J.M. Zeegers, 1968, Scand. J. Hematol. 5,179. Bernier, G.M., V. Tominaga, C.W. Easley and F.W. Putnam, 1965, Biochemistry 9, 2072. Fine, J.M. and M. Steinbuch, 1970, Rev. Eur. Etud. Clin. Biol. 15, 1115. Gold, E.R. and H.H. Fudenberg, 1967, J. Immunol. 99,859. Grabar, P. and C.A. Williams, 1953, Biochim. Biophys. Acta 10,193. Lubec, G., 1976, Pathology 4,526. Mehta, S.K., A.G. Plaut, N.J. Calvanico and T.B. Tomasi, 1973, J. Immunol. 111, 1274. Plaut, A.G., R.J. Genco and T.B. Tomasi, 1974, J. Immunol. 113,289. Plaut, A.G., J.V. Gilbert, M.J. Artenstein and J.D. Capra, 1975, Science 190, 1103. Shuster, J., 1971, Immunochemistry 8,405. Summers, D.T., J.V. Maizel and J.E. Darrell, 1965, Proc. Nat. Acad. Sci. U.S.A. 59,505. Virella, G., R. Valadas-Preto and F. Graca, 1975, Brit. J. Haematol. 30,479.