Immunology Letters, 31 (1992) 259- 266 Elsevier IMLET 01739
Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus Ludmila Proke~ovfi I , B~la Potu~.nikov~d, Jan P o t e m p a 2, Ji~i Zikgm3, Jill Radl 4, Luba Hachov~t5, Krystyna Baran 2, Zofia Porwit-BObr 2 and Ctirad John 1 ILaboratory of Special Medical Microbiology and Immunology, 1st Medical Faculty, Charles University, Studni6kova 7, Prague, Czechoslovakia, 2Institute of Molecular Biology, Jagiellonian University, AL Mickiewicza 3, Cracow, Poland, 3Institute of Microbiology, Czechoslovak Academy of Sciences, V(det~skd 1083, Prague, Czechoslovakia, 4TNO Institute for Experimental Gerontology, P.O. Box 5815, R(]swijk, The Netherlands, and 5SEVAC, Korunn/108, Prague, Czechoslovakia (Received 3 October 1991; accepted 15 October 1991)
1. Summary
The serine proteinase (SP) released into the environment by most strains of S. aureus cleaves human IgG, IgM and IgA of both subclasses - IgA 1 and IgA 2. SP cleaves H chains of all immunoglobulin classes and the SC of S-IgA, the L chains are degraded partially. The SP-induced cleavage results in a large spectrum of fragments under reducing conditions within a broad range of M r (approx. 41000 to < 12400). This indicates that the enzyme does not affect the Ig molecule in the hinge region only. The degree of cleavage depends on the enzyme:substrate ratio and on the duration of incubation. The generation of small fragments is associated with the loss of antigenic determinants that results from the decreased binding of the cleaved material in the ELISA method. Partial cleavage of L chains suggests that the enzyme alters part of the molecule that is involved in antigen binding. Even if the ability of antigen binding remains preserved after cleaving Ig with SP, the antibody function is disturbed by splitting off the Fc region or by its degradation into small fragments. SP has to be considered as one of the virulence factors of S. aureus Key words: Human immunoglobulin; Serine proteinase; Staphylococcus aureus; Cleavage Correspondence to: L. Proke~ovgt, Laboratory of Special Medical Microbiology and Immunology, 1st Medical Faculty, Charles University, Studni~kova 7, 12800 Prague 2, Czechoslovakia.
that may protect bacteria against the defence mechanisms of the host. 2. Introduction
A number of bacteria, playing an important role in human pathology, form proteinases that can cleave Ig of the host. This ability may be considered to be one of virulence factors impairing the immunological defence mechanisms of the macroorganism. Most studies on bacterial proteinases have been devoted to the cleavage of IgA. IgA plays an important role in the defence of mucosal surfaces that represent the site of invasion of many bacterial infections. Metha et al. [1] were the first to mention the existence of bacterial IgA-cleaving proteinases. Bacterial proteinases cleave mostly IgA 1 and do not cleave IgA 2. Such proteinases were described in pathogenic species of Haemophilus and Neisseria, in some streptococcal species, in Bacteroides, Capnocytophaga, Ureaplasma and Serratia marcescens [2, 3, 4]. Cleavage of IgA 1 occurs mostly in the hinge region. Proteinases cleaving both IgA subclasses have been described until now in Bacteroides gingivalis, Bacteroides intermedius [3], Clostridium ramosum [5] and in Pseudomonas aeruginosa [6]. Cleavage of other Ig classes has been described rarely. IgG-cleaving proteinase is formed by P. aeruginosa [7] and S. marcescens [4]. S. aureus is an important human pathogen. It forms three exoproteinases; a serine proteinase (SP), metalloproteinase and thiol proteinase [8]
that participate probably in the pathogenesis of staphylococcal infections. In 1983, cleavage of the rat IgG by staphylococcal SP was described [9]. Preliminary results concerning cleavage of human IgG, IgM and IgA obtained in our laboratory, were published in 1988 [10]. The aim of the present study is to further characterise the effect of staphylococcal SP on human Ig. 3. Materials and Methods
3.1. Serine proteinase SP produced by S. aureus strain V-8 was isolated according to Drapeau [11] using (NH4)2SO4 fractionation, acetone precipitation and DEAE-cellulose chromatography. The final purification included CM-Sephadex chromatography and gel filtration on Sephadex G-75. The SP obtained was homogeneous on SDS-PAGE. 3.2. Immunoglobulins IgG was isolated from the normal human serum by ethanol fractionation and precipitation with (NH4)2SO 4. Residues of IgA were removed by aluminium hydroxide precipitation [12]. IgM was isolated from the normal human serum by euglobulin precipitation by means of boric acid followed by gel filtration [13]. Secretory IgA (S-IgA) was isolated from human colostrum by ammonium sulphate precipitation and gel filtration [14]. IgA 2 (Fel) was isolated from plasma of a patient with IgA multiple myeloma as described previously [15]. The myeloma protein IgA 2-X was of A2m(2) allotype, mostly polymeric. All preparations were immunoelectrophoretically pure. 3.3. Cleavage of immunoglobulins by serine proteinase Cleavage was performed in a 0.02 M sodium phosphate buffer with 0.15 M NaC1, pH 7.8. The Ig concentration used was 1 mg/ml, the enzyme was present in various concentrations so that the enzyme:substrate ratios were 1:5 (I), 1:20 (II) or 1:100 (III). Ig was incubated together with the enzyme at 37 °C for 4 or 24 h. Ig incubated in the absence of the enzyme served as control (C). 260
3.4. SDS-PAGE An equal volume of twice concentrated denaturing buffer (0.062 M Tris hydrochloride, pH 6.8 with 2% SDS, 10% glycerol, and 5% 2-ME) was added to aliquots of samples after incubation and the samples were heated in a boiling water bath for 5 min. SDS-PAGE in 10% gel and in gradient gel ( 5 - 2 0 % ) was performed using the modification according to Laemmli [16]. For determination of protein molecular weight standards, Kit MS II (Serva) was used. After electrophoresis the gels were stained with Coomassie blue. 3.5. ELISA ELISA was performed in polystyrene microtitration plates (Koh-i-noor, Hardtmuth, Czechoslovakia) using the modification described by Tlaskalov~iet al. [17]. For Ig determination the plates were coated with anti-Ig antibodies (immunoglobulin fractions of pig antisera against the Fc fragment of human IgG, IgM and IgA: Q-SwAHu-IgG, QSwAHu-IgM and Q-SwAHu-IgA, SEVAC, Prague). For determination of antistaphylococcal antibodies the plates were coated with sonicate of S. aureus, strain Wood 46 (i.e. the strain which does not form the protein A). Diisopropylfluorophosphate (DFP) was added to aliquots of samples immediately after incubation, the final concentration in the sample being 1 mM. Such a treatment stopped the enzymatic reaction and prevented the potential cleavage of proteins coating the plates. DFP was kindly donated by Prof. J. Prokeg, Charles University, Prague. The binding of Ig or antistaphylococcal antibodies to the coated plate was determined by peroxidase-labelled pig antibodies against Fc fragments of individual human Ig (SwAHu-IgG/Px, SwAHu-IgM/Px and SwAHuIgA/Px, SEVAC, Prague). In one case, even monoclonal antibodies against IgA 1 and IgA 2 (NI 69 and NI 512, Nordic Immunological Laboratories Tilburg, The Netherlands) were used, followed by subsequent binding of pig anti-murine Ig labelled with peroxidase (SwAM/Px, SEVAC). The enzyme reaction was developed by o-phenylenediamine + H202, optical density of the coloured product was measured at 490 nm. Concentration of Ig and antibodies (in ng/ml) was read from the calibration
marker used (i.e. 12400), were generated. Thickening of the band in the region of L chains could be observed which was obviously caused by the fragments of 3' chains. The presence of 3' chain fragments in this region prevents the possibility to consider whether cleavage of L chains took place at the same time. During the cleavage of IgM (Fig. 2), a similar spectrum of fragments to IgG was generated. Contrary to IgG, weakening of the L chain band could be observed. As regards to the cleavage of S-IgA (Figs. 3 and 4), the ~ chain and the secretory component (SC) disappeared completely. Two larger fragments of M r 41000 and 27000 must be the products of degradation of these two S-IgA subunits. The weakening of L chain band suggests partial cleavage of the L chains. Similarly as with IgG and IgM a number of smaller split-products appeared. Cleavage of myeloma IgA 2 (Fig. 5) resulted in complete disappearance of not only a, but also L chains from the electrophoretic pattern. Otherwise a similar spectrum of fragments as with S-IgA can be seen. Thus, weakening or disappearance of L chain bands is visible in IgM and IgA only. As the L chains are identical in all Ig classes, one can assume that they are cleaved even in IgG. When Ig of all three classes are cleaved at lower enzyme: substrate ratios (II and III), the degrada-
curve for Ig giving the dependence of the optical density on the dilution of standard serum with known IgG, IgM and IgA concentration. 4. Results Serum IgG and IgM, colostral S-IgA and myeloma IgA 2 were incubated in the presence of SP for 4 or 24 h at various enzyme:substrate ratios. The fate of Ig after incubation with the enzyme was followed both by SDS-PAGE and immunoenzyme assay (ELISA) in order to obtain data about the M r and immunochemical properties of proteolytic fragments. 4.1. Determination of Ig cleavage by means of
SDS-PAGE SDS-PAGE performed under reducing conditions clearly demonstrated cleavage of IgG, IgM and IgA with SP (Figs. 1,2, 3 and 4). The degree of cleavage is dependent on the enzyme:substrate ratio. At 1:5 ratio (I) the complete disappearance of the H chain band takes place in all three Ig classes after a 4-h incubation. During the cleavage of IgG (Fig. 1) a wide spectrum of fragments, with an M r of approx. 28 000 to lower than that of the smallest
67 kDa 45 kDa,.@
12,4kDa
-,,)
1
2
3
4
5
6
7
8
9
~8
Fig. 1. SDS-PAGE run in reducing conditions in gradient gel. Serum IgG after incubation with SP. Lanes 1 and 6, protein Mr standards; lanes 2 and 7, IgG incubated without enzyme; lanes 3 and 8, enzyme:substrateratio = 1:5; lanes 4 and 9, enzyme:substrate ratio = 1:20; lanes 5 and 10, enzyme:substrateratio = l: 100. Lanes 2, 3, 4 and 5, 4-h incubation; lanes 7, 8, 9 and 10, 24-h incubation. 261
•-,)
m
~
SP L
"=')
N
1
2
--67 kDa 1
45
i,,I,
12 ,,,,4 kDa
kOa
--N
3
4
5
6
7
8
Fig. 2. SDS-PAGE run under reducing conditions in gradient gel. Serum IgM after incubation with SP. Lanes 1 and 5, IgM incubated without enzyme; lanes 2 and 6, enzyme:substrate ratio = 1:5; lanes 3 and 7, enzyme:substrate ratio = 1:20; lanes 4 and 8, enzyme'substrate ratio = 1:100. Lanes 1, 2, 3 and 4, 4-h incubation; lanes 5, 6, 7 and 8, 24-h incubation.
SC &
SP L
Fig. 3. SDS-PAGE run under reducing conditions in 10% gel. S-IgA after incubation with SP 4 h. Lane 1, S-IgA incubated without enzyme; lanes 2, 3 and 4, enzyme:substrate ratio = 1:5, 1:20 and 1:100, respectively; lane 5, SP after 4-h incubation; lanes 6, 7, 8 and 9, protein M r standards.
tion is less intensive but still evident. The pattern of proteolytic fragments obtained after a 24-h incubation of Ig with SP is similar as that after a 4-h incubation; however, the number of small split-products was slightly increased.
262
The enzyme alone formed row bands located closely to of the band might be caused cleavage which may occur in
in SDS-PAGE 2 nareach other. Doubling by a partial autolytic SP [8].
I
2
3
4
5
6
7
8
Fig. 4. SDS-PAGE run under reducing conditions in gradient gel. S-IgA after incubation with SP. Lanes 1 and 5, S-IgA incubated without enzyme; lanes 2 and 6, enzyme:substrate ratio = 1:5; lanes 3 and 7, enzyme:substrate ratio = 1:20; lanes 4 and 8, enzyme:substrate ratio = h i 0 0 . Lanes i, 2, 3 and 4, 4-h incubation; lanes 5, 6, 7 and 8, 24-h incubation.
•- - 6 7
kDo
--45kDa
decrease of Ig detectable by the ELISA method after cleavage with SP. The results are expressed as the percentage of the control incubated under identical conditions in the absence of the enzyme. Since antibodies against the Fc fragment were used for the determination of Ig, the decrease of Ig values after cleavage with SP indicates the loss of antigenic determinants in this part of the molecule. It follows that part of Fc is degraded into small fragments under the influence of the enzyme. With respect to the three Ig classes that were determined by three polyclonal antibodies, we were unable to compare quantitatively the results of cleavage of
=i? TABLE 1 Fig. 5. SDS-PAGE run under reducing conditions in gradient gel. Myeloma IgA 2-X after incubation with SP 4 h. Lane l, IgA 2 incubated without enzyme; lanes 2, 3 and 4, enzyme:substrate ratio = 1:5, 1:20 and h i 0 0 , respectively.
ELISA detection of Ig after 4-h and 24-h incubation with serine proteinase of S. aureus. Results are expressed in percent (Ig concentration in the control incubated in the absence o f the enzyme = 100%).
4.2. Determination of lg cleavage by the ELISA
method Unsplit and enzymatically cleaved Ig were determined as antigens using plates coated with antibodies against individual Ig classes. Table 1 shows a
C I II III
Enzyme substrate ratio
IgG
IgM
S-IgA
4 h
24 h
4 h
24 h
4 h
24 h
0 1:5 1:20 1:100
100 70 72 90
100 38 52 62
100 78 79 93
100 76 70 75
100 86 93 104
100 78 91 91
263
individual classes. Nevertheless, one can assume, particularly on the basis of the time dependence of changes (i.e. comparison of results obtained after 4-h and 24-h cleavage), that the Fc region of IgG is the most sensitive to the action of SP. In the second run of the ELISA test, Ig was determined as an antibody (Table 2). All three classes of human Ig contain a sufficient proportion of antibodies against S. a u r e u s . Therefore we tested changes of antibodies after incubation with SP by detecting antibodies against staphylococci. Using the plates coated with sonicate of S. a u r e u s we determined either the whole antibody molecules or the fragments, possessing the antibody combining site and at least a part of the Fc, because the conjugates used for the determination of the antibody binding to the plate contained antibodies against the Fc part of the Ig molecule only. A high susceptibility of IgG to the effect of SP could again be observed: in most antibody molecules disturbance of the antibody combining site or splitting off of the Fc occurred after 24-h incubation with the enzyme. S-IgA was less susceptible. With regard to cleaved S-IgA, binding of antibody to antigen was also determined by means of monoclonal antibodies against Fc of IgA 1 and IgA 2. These results are not included in the table containing the means of several determinations, because the experiment was performed only once. At the enzyme:substrate ratio 1:5 the amount of detectable IgA 1 and IgA 2 antibodies decreased to 47°7o and 68%, respectively, after 4-h incubation. It proves that SP cleaves both IgA 1 and IgA 2. When IgM was determined as an antibody, no significant changes occurred after incubation with SP, although, as can be seen in TABLE 2 ELISA binding of Ig incubated 4 h and 24 h with serine proteinase to antigens of S. aureus. Results are expressed in percent (Ig amount of the uncleaved control that binds with S. a u r e u s sonicate = 100°70).
C I II III
264
Enzyme substrate ratio
IgG
IgM
S-IgA
4 h
24 h
4 h
24 h
4 h
24 h
0 1:5 1:20 1:100
100 28 64 98
100 4 10 36
100 92 96 120
100 104 90 98
100 39 60 64
100 36 47 63
Table 1, the Fc of IgM may assume that most contain at least a part bound by antibodies of
was partially cleaved. One antigen binding fragments of the Fc region which is the conjugate.
5. Discussion
SP is formed by at least 67% of S. a u r e u s [8]. Therefore, its potential participation in the pathogenesis of most diseases caused by this microorganism must be taken into consideration. Besides the possible direct proteolytic effect on tissues during infection it increases tissue degradation by macroorganism proteinases by inactivating the plasma 1-proteinase inhibitor [18]. The immune reactions can be negatively influenced through the inhibitory effect of SP on polyclonal activation of B cells [10]. Thus, cleavage of Ig by SP represents another factor negatively influencing the defence mechanisms of the host. Our experiments have shown that the SP of S. a u r e u s cleaves all three main Ig classes. Analysis of cleaved Ig by SDS-PAGE revealed the complete degradation of H chains of all three classes while the L chains were affected partially. The results of our experiments do not allow us to conclude whether Fab and Fc fragments are generated during cleavage. However, the split-products of 7 chains causing thickening of the band in the region of L chains might include parts of Fab and Fc released after reduction of the disulphide bonds. Partial cleavage of L chains suggests that the enzyme attacks even the Fab portion of the molecule, which is responsible for the antigen binding. Cleavage of L chains and the presence of a large spectrum of fragments of various M r proves that SP acts on multiple sites of the molecule and not on the hinge region only. As to IgA, we proved cleavage of both S-IgA and serum myeloma IgA 2-X. SP cleaves o~and L chains and even the SC. Myeloma IgA 1 was not available; however, complete disappearance of the a chain in the electrophoretic pattern of cleaved S-IgA isolated from the colostrum, where IgA 1 is present in a higher proportion than IgA 2, serves as sufficient proof that SP cleaves not only IgA 2 but also IgA 1. Complete disappearance of the band of L chains after cleaving the myeloma IgA 2-X and weakening of the L chain band after cleaving poly-
clonal IgM and S-IgA, might suggest that SP cleaves ~ chains and does not cleave ct chains. One may conclude that contrary to most of the so far described Ig cleaving, bacterial proteinases belonging mostly to metalloproteinases [3, 4, 6], the staphylococcal serine proteinase cleaves not only in the hinge region but also in other parts of the molecule and, in addition, cleaves both IgA 1 and IgA 2. It also cleaves IgG and IgM, the cleavage of which by other bacterial enzymes has not been systematically studied so far. The generation of a large number of small fragments after SP cleavage of Ig, that is probably accompanied by the loss of some antigenic determinants, is manifested by decreased possibility to determine cleaved Ig by means of the ELISA. When determining Ig as an antigen or as an antibody, we have shown that the consequences of cleaving by SP were different in individual Ig classes. The most sensitive class was found to be IgG. The least significant effect of SP cleavage was in IgM when tested as an antibody. We have even found a slight increase in the ability of antigen binding (Table 2). This might be caused by decreased steric hindrance during the antigen-antibody reaction that can occur after partial proteolysis of the large IgM molecule. Although a certain proportion of Ig molecules of all classes preserved the ability of antigen binding after SP cleavage, it does not mean the antibody molecules are fully functional. In all Ig classes cleaving of all H chains takes place at the highest enzyme:substrate ratio and even with a lower amount of enzyme part of the H chains is cleaved. Thus, fragments possessing the ability of antigen binding cannot contain intact Fc that mediates functions important for the defence against infection. Cleavage of main three Ig classes by staphylococcal SP may protect bacteria against antibodies not only on the mucous membranes but anywhere in the body and therefore SP appears to be one of the important virulence factors of S. a u r e u s .
Acknowledgements The authors thank Mrs E. Formfinkovfi and Mrs M. gafrfinkovfi for skillful technical assistance.
References [1] Metha, S. K., Plaut, A. G., Calvanico, N. J. and Tomasi, Jr., T. B. (1973) J. Immunol. 111, 1274. [2] Plaut, A. G., Wistar, Jr., R. and Capra, J. D. (1974) J. Clin. Invest. 54, 1295. [3] Kilian, M., Thomsen, B., Petersen, T. E. and Bleeg, H. S. (1983) Ann. NY Acad. Sci. 409, 612. [4] Molla, A., Kagimito, T. and Maeda, H. (1988) Infect. Immun. 56, 916.
[5] Fujiyama, Y., Kobayashi,K., Senda, S., Benno, Y., Bamba, T. and Hosoda, S. (1985) J. Immunol. 134, 573. [6] Heck, L. W., Alarcon, P. G., Kulhavy, R. M., Morihara, K., Russell, M. W. and Mestecky, J. F. (1990) J. Immunol. 144, 2253. [7] D6ring, G., Obernesser, H.-J. and Botzenhart, K. (1981) Zentral. Bakteriol. Mikrobiol. Hyg. 249, 89. [8] Arvidson,S. D. (1983)in: Staphylococciand Staphylococcal Infections (C. S. F. Easmon and C. Adlam, Eds.) Vol. 2, pp. 780- 786, AcademicPress, London. [9] Rousseaux, J., Rousseaux-Pr6vost, R., Bazin, H. and Biserte, G. (1983) Biochim. Biophys. Acta 748,205. [10] Prokegovfi,L., Porwit-B6br, Z., Baran, K., Potempa, J. and John, C. (1988) lmmunol. Lett. 19, 127. [11] Drapeau, G. R. (1978) Can. J. Biochem. 56, 534. [12] Penell, R.B. (1960) in: The Plasma Proteins (F. W. Putnam, Ed.) Vol. I, pp. 9-50, Academic Press, New York. [13] Masseyeff,R., Gombert, J. and Josselin, J. (1965) Immunochemistry 2, 177. [14] Zik~in, J., Mestecky, J., Kulhavy, R. and Bennett, J. C. (1986) Mol. Immunol. 23,541. [15] Mestecky,J., Schrohenloher, R. E., Kulhavy, R., Wright, G. P. and Tomana, M. (1974) Proc. Natl. Acad. Sci. USA 71,544. [16] Laemmli,U. K. (1970) Nature 227,680. [17] Tlaskalovfi-Hogenovfi, H., Bfirtov~i, J., Mrklas, L., ManUal, P., Broukal, Z., Barot-Ciorbaru, R., Movfik, M. and Hanik~,fovfi,M. (1985) Folia Microbiol. 30, 258. [18] Potempa, J., Watorek, W. and Travis, J. (1986) J. Biol. Chem. 261, 14330.
265
Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus Ludmila Proke~ovfi I , B~la Potu~.nikov~d, Jan P o t e m p a 2, Ji~i Zikgm3, Jill Radl 4, Luba Hachov~t5, Krystyna Baran 2, Zofia Porwit-BObr 2 and Ctirad John 1 ILaboratory of Special Medical Microbiology and Immunology, 1st Medical Faculty, Charles University, Studni6kova 7, Prague, Czechoslovakia, 2Institute of Molecular Biology, Jagiellonian University, AL Mickiewicza 3, Cracow, Poland, 3Institute of Microbiology, Czechoslovak Academy of Sciences, V(det~skd 1083, Prague, Czechoslovakia, 4TNO Institute for Experimental Gerontology, P.O. Box 5815, R(]swijk, The Netherlands, and 5SEVAC, Korunn/108, Prague, Czechoslovakia (Received 3 October 1991; accepted 15 October 1991)
1. Summary
The serine proteinase (SP) released into the environment by most strains of S. aureus cleaves human IgG, IgM and IgA of both subclasses - IgA 1 and IgA 2. SP cleaves H chains of all immunoglobulin classes and the SC of S-IgA, the L chains are degraded partially. The SP-induced cleavage results in a large spectrum of fragments under reducing conditions within a broad range of M r (approx. 41000 to < 12400). This indicates that the enzyme does not affect the Ig molecule in the hinge region only. The degree of cleavage depends on the enzyme:substrate ratio and on the duration of incubation. The generation of small fragments is associated with the loss of antigenic determinants that results from the decreased binding of the cleaved material in the ELISA method. Partial cleavage of L chains suggests that the enzyme alters part of the molecule that is involved in antigen binding. Even if the ability of antigen binding remains preserved after cleaving Ig with SP, the antibody function is disturbed by splitting off the Fc region or by its degradation into small fragments. SP has to be considered as one of the virulence factors of S. aureus Key words: Human immunoglobulin; Serine proteinase; Staphylococcus aureus; Cleavage Correspondence to: L. Proke~ovgt, Laboratory of Special Medical Microbiology and Immunology, 1st Medical Faculty, Charles University, Studni~kova 7, 12800 Prague 2, Czechoslovakia.
that may protect bacteria against the defence mechanisms of the host. 2. Introduction
A number of bacteria, playing an important role in human pathology, form proteinases that can cleave Ig of the host. This ability may be considered to be one of virulence factors impairing the immunological defence mechanisms of the macroorganism. Most studies on bacterial proteinases have been devoted to the cleavage of IgA. IgA plays an important role in the defence of mucosal surfaces that represent the site of invasion of many bacterial infections. Metha et al. [1] were the first to mention the existence of bacterial IgA-cleaving proteinases. Bacterial proteinases cleave mostly IgA 1 and do not cleave IgA 2. Such proteinases were described in pathogenic species of Haemophilus and Neisseria, in some streptococcal species, in Bacteroides, Capnocytophaga, Ureaplasma and Serratia marcescens [2, 3, 4]. Cleavage of IgA 1 occurs mostly in the hinge region. Proteinases cleaving both IgA subclasses have been described until now in Bacteroides gingivalis, Bacteroides intermedius [3], Clostridium ramosum [5] and in Pseudomonas aeruginosa [6]. Cleavage of other Ig classes has been described rarely. IgG-cleaving proteinase is formed by P. aeruginosa [7] and S. marcescens [4]. S. aureus is an important human pathogen. It forms three exoproteinases; a serine proteinase (SP), metalloproteinase and thiol proteinase [8]
that participate probably in the pathogenesis of staphylococcal infections. In 1983, cleavage of the rat IgG by staphylococcal SP was described [9]. Preliminary results concerning cleavage of human IgG, IgM and IgA obtained in our laboratory, were published in 1988 [10]. The aim of the present study is to further characterise the effect of staphylococcal SP on human Ig. 3. Materials and Methods
3.1. Serine proteinase SP produced by S. aureus strain V-8 was isolated according to Drapeau [11] using (NH4)2SO4 fractionation, acetone precipitation and DEAE-cellulose chromatography. The final purification included CM-Sephadex chromatography and gel filtration on Sephadex G-75. The SP obtained was homogeneous on SDS-PAGE. 3.2. Immunoglobulins IgG was isolated from the normal human serum by ethanol fractionation and precipitation with (NH4)2SO 4. Residues of IgA were removed by aluminium hydroxide precipitation [12]. IgM was isolated from the normal human serum by euglobulin precipitation by means of boric acid followed by gel filtration [13]. Secretory IgA (S-IgA) was isolated from human colostrum by ammonium sulphate precipitation and gel filtration [14]. IgA 2 (Fel) was isolated from plasma of a patient with IgA multiple myeloma as described previously [15]. The myeloma protein IgA 2-X was of A2m(2) allotype, mostly polymeric. All preparations were immunoelectrophoretically pure. 3.3. Cleavage of immunoglobulins by serine proteinase Cleavage was performed in a 0.02 M sodium phosphate buffer with 0.15 M NaC1, pH 7.8. The Ig concentration used was 1 mg/ml, the enzyme was present in various concentrations so that the enzyme:substrate ratios were 1:5 (I), 1:20 (II) or 1:100 (III). Ig was incubated together with the enzyme at 37 °C for 4 or 24 h. Ig incubated in the absence of the enzyme served as control (C). 260
3.4. SDS-PAGE An equal volume of twice concentrated denaturing buffer (0.062 M Tris hydrochloride, pH 6.8 with 2% SDS, 10% glycerol, and 5% 2-ME) was added to aliquots of samples after incubation and the samples were heated in a boiling water bath for 5 min. SDS-PAGE in 10% gel and in gradient gel ( 5 - 2 0 % ) was performed using the modification according to Laemmli [16]. For determination of protein molecular weight standards, Kit MS II (Serva) was used. After electrophoresis the gels were stained with Coomassie blue. 3.5. ELISA ELISA was performed in polystyrene microtitration plates (Koh-i-noor, Hardtmuth, Czechoslovakia) using the modification described by Tlaskalov~iet al. [17]. For Ig determination the plates were coated with anti-Ig antibodies (immunoglobulin fractions of pig antisera against the Fc fragment of human IgG, IgM and IgA: Q-SwAHu-IgG, QSwAHu-IgM and Q-SwAHu-IgA, SEVAC, Prague). For determination of antistaphylococcal antibodies the plates were coated with sonicate of S. aureus, strain Wood 46 (i.e. the strain which does not form the protein A). Diisopropylfluorophosphate (DFP) was added to aliquots of samples immediately after incubation, the final concentration in the sample being 1 mM. Such a treatment stopped the enzymatic reaction and prevented the potential cleavage of proteins coating the plates. DFP was kindly donated by Prof. J. Prokeg, Charles University, Prague. The binding of Ig or antistaphylococcal antibodies to the coated plate was determined by peroxidase-labelled pig antibodies against Fc fragments of individual human Ig (SwAHu-IgG/Px, SwAHu-IgM/Px and SwAHuIgA/Px, SEVAC, Prague). In one case, even monoclonal antibodies against IgA 1 and IgA 2 (NI 69 and NI 512, Nordic Immunological Laboratories Tilburg, The Netherlands) were used, followed by subsequent binding of pig anti-murine Ig labelled with peroxidase (SwAM/Px, SEVAC). The enzyme reaction was developed by o-phenylenediamine + H202, optical density of the coloured product was measured at 490 nm. Concentration of Ig and antibodies (in ng/ml) was read from the calibration
marker used (i.e. 12400), were generated. Thickening of the band in the region of L chains could be observed which was obviously caused by the fragments of 3' chains. The presence of 3' chain fragments in this region prevents the possibility to consider whether cleavage of L chains took place at the same time. During the cleavage of IgM (Fig. 2), a similar spectrum of fragments to IgG was generated. Contrary to IgG, weakening of the L chain band could be observed. As regards to the cleavage of S-IgA (Figs. 3 and 4), the ~ chain and the secretory component (SC) disappeared completely. Two larger fragments of M r 41000 and 27000 must be the products of degradation of these two S-IgA subunits. The weakening of L chain band suggests partial cleavage of the L chains. Similarly as with IgG and IgM a number of smaller split-products appeared. Cleavage of myeloma IgA 2 (Fig. 5) resulted in complete disappearance of not only a, but also L chains from the electrophoretic pattern. Otherwise a similar spectrum of fragments as with S-IgA can be seen. Thus, weakening or disappearance of L chain bands is visible in IgM and IgA only. As the L chains are identical in all Ig classes, one can assume that they are cleaved even in IgG. When Ig of all three classes are cleaved at lower enzyme: substrate ratios (II and III), the degrada-
curve for Ig giving the dependence of the optical density on the dilution of standard serum with known IgG, IgM and IgA concentration. 4. Results Serum IgG and IgM, colostral S-IgA and myeloma IgA 2 were incubated in the presence of SP for 4 or 24 h at various enzyme:substrate ratios. The fate of Ig after incubation with the enzyme was followed both by SDS-PAGE and immunoenzyme assay (ELISA) in order to obtain data about the M r and immunochemical properties of proteolytic fragments. 4.1. Determination of Ig cleavage by means of
SDS-PAGE SDS-PAGE performed under reducing conditions clearly demonstrated cleavage of IgG, IgM and IgA with SP (Figs. 1,2, 3 and 4). The degree of cleavage is dependent on the enzyme:substrate ratio. At 1:5 ratio (I) the complete disappearance of the H chain band takes place in all three Ig classes after a 4-h incubation. During the cleavage of IgG (Fig. 1) a wide spectrum of fragments, with an M r of approx. 28 000 to lower than that of the smallest
67 kDa 45 kDa,.@
12,4kDa
-,,)
1
2
3
4
5
6
7
8
9
~8
Fig. 1. SDS-PAGE run in reducing conditions in gradient gel. Serum IgG after incubation with SP. Lanes 1 and 6, protein Mr standards; lanes 2 and 7, IgG incubated without enzyme; lanes 3 and 8, enzyme:substrateratio = 1:5; lanes 4 and 9, enzyme:substrate ratio = 1:20; lanes 5 and 10, enzyme:substrateratio = l: 100. Lanes 2, 3, 4 and 5, 4-h incubation; lanes 7, 8, 9 and 10, 24-h incubation. 261
•-,)
m
~
SP L
"=')
N
1
2
--67 kDa 1
45
i,,I,
12 ,,,,4 kDa
kOa
--N
3
4
5
6
7
8
Fig. 2. SDS-PAGE run under reducing conditions in gradient gel. Serum IgM after incubation with SP. Lanes 1 and 5, IgM incubated without enzyme; lanes 2 and 6, enzyme:substrate ratio = 1:5; lanes 3 and 7, enzyme:substrate ratio = 1:20; lanes 4 and 8, enzyme'substrate ratio = 1:100. Lanes 1, 2, 3 and 4, 4-h incubation; lanes 5, 6, 7 and 8, 24-h incubation.
SC &
SP L
Fig. 3. SDS-PAGE run under reducing conditions in 10% gel. S-IgA after incubation with SP 4 h. Lane 1, S-IgA incubated without enzyme; lanes 2, 3 and 4, enzyme:substrate ratio = 1:5, 1:20 and 1:100, respectively; lane 5, SP after 4-h incubation; lanes 6, 7, 8 and 9, protein M r standards.
tion is less intensive but still evident. The pattern of proteolytic fragments obtained after a 24-h incubation of Ig with SP is similar as that after a 4-h incubation; however, the number of small split-products was slightly increased.
262
The enzyme alone formed row bands located closely to of the band might be caused cleavage which may occur in
in SDS-PAGE 2 nareach other. Doubling by a partial autolytic SP [8].
I
2
3
4
5
6
7
8
Fig. 4. SDS-PAGE run under reducing conditions in gradient gel. S-IgA after incubation with SP. Lanes 1 and 5, S-IgA incubated without enzyme; lanes 2 and 6, enzyme:substrate ratio = 1:5; lanes 3 and 7, enzyme:substrate ratio = 1:20; lanes 4 and 8, enzyme:substrate ratio = h i 0 0 . Lanes i, 2, 3 and 4, 4-h incubation; lanes 5, 6, 7 and 8, 24-h incubation.
•- - 6 7
kDo
--45kDa
decrease of Ig detectable by the ELISA method after cleavage with SP. The results are expressed as the percentage of the control incubated under identical conditions in the absence of the enzyme. Since antibodies against the Fc fragment were used for the determination of Ig, the decrease of Ig values after cleavage with SP indicates the loss of antigenic determinants in this part of the molecule. It follows that part of Fc is degraded into small fragments under the influence of the enzyme. With respect to the three Ig classes that were determined by three polyclonal antibodies, we were unable to compare quantitatively the results of cleavage of
=i? TABLE 1 Fig. 5. SDS-PAGE run under reducing conditions in gradient gel. Myeloma IgA 2-X after incubation with SP 4 h. Lane l, IgA 2 incubated without enzyme; lanes 2, 3 and 4, enzyme:substrate ratio = 1:5, 1:20 and h i 0 0 , respectively.
ELISA detection of Ig after 4-h and 24-h incubation with serine proteinase of S. aureus. Results are expressed in percent (Ig concentration in the control incubated in the absence o f the enzyme = 100%).
4.2. Determination of lg cleavage by the ELISA
method Unsplit and enzymatically cleaved Ig were determined as antigens using plates coated with antibodies against individual Ig classes. Table 1 shows a
C I II III
Enzyme substrate ratio
IgG
IgM
S-IgA
4 h
24 h
4 h
24 h
4 h
24 h
0 1:5 1:20 1:100
100 70 72 90
100 38 52 62
100 78 79 93
100 76 70 75
100 86 93 104
100 78 91 91
263
individual classes. Nevertheless, one can assume, particularly on the basis of the time dependence of changes (i.e. comparison of results obtained after 4-h and 24-h cleavage), that the Fc region of IgG is the most sensitive to the action of SP. In the second run of the ELISA test, Ig was determined as an antibody (Table 2). All three classes of human Ig contain a sufficient proportion of antibodies against S. a u r e u s . Therefore we tested changes of antibodies after incubation with SP by detecting antibodies against staphylococci. Using the plates coated with sonicate of S. a u r e u s we determined either the whole antibody molecules or the fragments, possessing the antibody combining site and at least a part of the Fc, because the conjugates used for the determination of the antibody binding to the plate contained antibodies against the Fc part of the Ig molecule only. A high susceptibility of IgG to the effect of SP could again be observed: in most antibody molecules disturbance of the antibody combining site or splitting off of the Fc occurred after 24-h incubation with the enzyme. S-IgA was less susceptible. With regard to cleaved S-IgA, binding of antibody to antigen was also determined by means of monoclonal antibodies against Fc of IgA 1 and IgA 2. These results are not included in the table containing the means of several determinations, because the experiment was performed only once. At the enzyme:substrate ratio 1:5 the amount of detectable IgA 1 and IgA 2 antibodies decreased to 47°7o and 68%, respectively, after 4-h incubation. It proves that SP cleaves both IgA 1 and IgA 2. When IgM was determined as an antibody, no significant changes occurred after incubation with SP, although, as can be seen in TABLE 2 ELISA binding of Ig incubated 4 h and 24 h with serine proteinase to antigens of S. aureus. Results are expressed in percent (Ig amount of the uncleaved control that binds with S. a u r e u s sonicate = 100°70).
C I II III
264
Enzyme substrate ratio
IgG
IgM
S-IgA
4 h
24 h
4 h
24 h
4 h
24 h
0 1:5 1:20 1:100
100 28 64 98
100 4 10 36
100 92 96 120
100 104 90 98
100 39 60 64
100 36 47 63
Table 1, the Fc of IgM may assume that most contain at least a part bound by antibodies of
was partially cleaved. One antigen binding fragments of the Fc region which is the conjugate.
5. Discussion
SP is formed by at least 67% of S. a u r e u s [8]. Therefore, its potential participation in the pathogenesis of most diseases caused by this microorganism must be taken into consideration. Besides the possible direct proteolytic effect on tissues during infection it increases tissue degradation by macroorganism proteinases by inactivating the plasma 1-proteinase inhibitor [18]. The immune reactions can be negatively influenced through the inhibitory effect of SP on polyclonal activation of B cells [10]. Thus, cleavage of Ig by SP represents another factor negatively influencing the defence mechanisms of the host. Our experiments have shown that the SP of S. a u r e u s cleaves all three main Ig classes. Analysis of cleaved Ig by SDS-PAGE revealed the complete degradation of H chains of all three classes while the L chains were affected partially. The results of our experiments do not allow us to conclude whether Fab and Fc fragments are generated during cleavage. However, the split-products of 7 chains causing thickening of the band in the region of L chains might include parts of Fab and Fc released after reduction of the disulphide bonds. Partial cleavage of L chains suggests that the enzyme attacks even the Fab portion of the molecule, which is responsible for the antigen binding. Cleavage of L chains and the presence of a large spectrum of fragments of various M r proves that SP acts on multiple sites of the molecule and not on the hinge region only. As to IgA, we proved cleavage of both S-IgA and serum myeloma IgA 2-X. SP cleaves o~and L chains and even the SC. Myeloma IgA 1 was not available; however, complete disappearance of the a chain in the electrophoretic pattern of cleaved S-IgA isolated from the colostrum, where IgA 1 is present in a higher proportion than IgA 2, serves as sufficient proof that SP cleaves not only IgA 2 but also IgA 1. Complete disappearance of the band of L chains after cleaving the myeloma IgA 2-X and weakening of the L chain band after cleaving poly-
clonal IgM and S-IgA, might suggest that SP cleaves ~ chains and does not cleave ct chains. One may conclude that contrary to most of the so far described Ig cleaving, bacterial proteinases belonging mostly to metalloproteinases [3, 4, 6], the staphylococcal serine proteinase cleaves not only in the hinge region but also in other parts of the molecule and, in addition, cleaves both IgA 1 and IgA 2. It also cleaves IgG and IgM, the cleavage of which by other bacterial enzymes has not been systematically studied so far. The generation of a large number of small fragments after SP cleavage of Ig, that is probably accompanied by the loss of some antigenic determinants, is manifested by decreased possibility to determine cleaved Ig by means of the ELISA. When determining Ig as an antigen or as an antibody, we have shown that the consequences of cleaving by SP were different in individual Ig classes. The most sensitive class was found to be IgG. The least significant effect of SP cleavage was in IgM when tested as an antibody. We have even found a slight increase in the ability of antigen binding (Table 2). This might be caused by decreased steric hindrance during the antigen-antibody reaction that can occur after partial proteolysis of the large IgM molecule. Although a certain proportion of Ig molecules of all classes preserved the ability of antigen binding after SP cleavage, it does not mean the antibody molecules are fully functional. In all Ig classes cleaving of all H chains takes place at the highest enzyme:substrate ratio and even with a lower amount of enzyme part of the H chains is cleaved. Thus, fragments possessing the ability of antigen binding cannot contain intact Fc that mediates functions important for the defence against infection. Cleavage of main three Ig classes by staphylococcal SP may protect bacteria against antibodies not only on the mucous membranes but anywhere in the body and therefore SP appears to be one of the important virulence factors of S. a u r e u s .
Acknowledgements The authors thank Mrs E. Formfinkovfi and Mrs M. gafrfinkovfi for skillful technical assistance.
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[5] Fujiyama, Y., Kobayashi,K., Senda, S., Benno, Y., Bamba, T. and Hosoda, S. (1985) J. Immunol. 134, 573. [6] Heck, L. W., Alarcon, P. G., Kulhavy, R. M., Morihara, K., Russell, M. W. and Mestecky, J. F. (1990) J. Immunol. 144, 2253. [7] D6ring, G., Obernesser, H.-J. and Botzenhart, K. (1981) Zentral. Bakteriol. Mikrobiol. Hyg. 249, 89. [8] Arvidson,S. D. (1983)in: Staphylococciand Staphylococcal Infections (C. S. F. Easmon and C. Adlam, Eds.) Vol. 2, pp. 780- 786, AcademicPress, London. [9] Rousseaux, J., Rousseaux-Pr6vost, R., Bazin, H. and Biserte, G. (1983) Biochim. Biophys. Acta 748,205. [10] Prokegovfi,L., Porwit-B6br, Z., Baran, K., Potempa, J. and John, C. (1988) lmmunol. Lett. 19, 127. [11] Drapeau, G. R. (1978) Can. J. Biochem. 56, 534. [12] Penell, R.B. (1960) in: The Plasma Proteins (F. W. Putnam, Ed.) Vol. I, pp. 9-50, Academic Press, New York. [13] Masseyeff,R., Gombert, J. and Josselin, J. (1965) Immunochemistry 2, 177. [14] Zik~in, J., Mestecky, J., Kulhavy, R. and Bennett, J. C. (1986) Mol. Immunol. 23,541. [15] Mestecky,J., Schrohenloher, R. E., Kulhavy, R., Wright, G. P. and Tomana, M. (1974) Proc. Natl. Acad. Sci. USA 71,544. [16] Laemmli,U. K. (1970) Nature 227,680. [17] Tlaskalovfi-Hogenovfi, H., Bfirtov~i, J., Mrklas, L., ManUal, P., Broukal, Z., Barot-Ciorbaru, R., Movfik, M. and Hanik~,fovfi,M. (1985) Folia Microbiol. 30, 258. [18] Potempa, J., Watorek, W. and Travis, J. (1986) J. Biol. Chem. 261, 14330.
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