Acta path. microbiol. immunol. scand. Sect. B, 94: 69-73, 1986.

TWO SEPARATE NON-IMMUNE INTERACTIONS BETWEEN STAPHYLOCOCCAL PROTEIN A

AND IMMUNOGLOBULINS ARE MEDIATED BY STRUCTURES ON y CHAINS

MATS ERNTELL’, ERLING B. MYHRE‘ and W R A N KRONVALL’

Departments of Infectious Diseases and Medical Microbiology, University Hospital, Lund, Sweden

Emtell, M., Myhre, E. B. & Kronvall, G. Two separate non-immune interactions between staphylococcal protein A and immunoglobulins are mediated by structures on y chains. Acta path. microbiol. immunol. scand. Sect. B, 94: 69-73, 1986.

The present investigation was undertaken to determine whether the light or the heavy immunoglobulin chain is involved in the alternative, non-immune F(ab’)2-mediated binding to staphylococcal protein A. Purified human polyclonal IgG was mildly reduced with dithiothreitol and alkylated with iodoacetamide. Intact IgG, purified light and heavy chains of polyclonal immunoglobulin G were tested in an inhibition assay for alternative non-immune F(ab)2-mediated binding to the protein A-carrying S. auras, strain Cowan I. The IgG Fc-mediated binding to protein A was studied in parallel inhibition experiments. Heavy chains inhibited both the alternative F(ab’)2- and the classical Fc-mediated binding to protein A. Isolated light chains were non-reactive. Intact IgC molecules were more potent inhibitors than isolated heavy chains tested in equimolar concentrations. Our results indicate that the alternative non-immune interaction between staphylococcal protein A and human immunoglobulins is mediated by structures expressed on the heavy immunoglobulin G chain. Thus, there are two separate protein A binding sites on y chains.

Key words: Protein A; y chains; non-immune binding.

Mats Erntell, Department of Infectious Diseases, University Hospital, S-221 85 Lund, Sweden.

Accepted as submitted 24.x.85 ments presented indicate that the heavy immuno- Staphylococcal protein A interacts in a non-im- globulin chain cames the reactive site. The light

mune way with the Fc part of human IgG (6). chain may, however, contribute to the reactivity, Another non-immune interaction between protein presumably by passive stabilization of the immu- A and the Fab portion of immunoglobulin A, E, G noglobulin molecule. and M has been described (3, 8). Inganas et al. showed that the Fc and F(ab’)2 interactions with protein A were independent of each other (10). Biguzzi located the Fab interaction to the Fv fragment ( 1 ) . He also described an interference Bacterial strains, growth and culture conditions. A pro- between the two types of protein A interactions, tein A-carrying SwWococcus auras strain, Cowan I

(NCTC no. 8530), was used as a protein A positive organism expressing both IgG Fc- and immunoglobulin suggesting that Fcy-like properties are expressed Fab-reactivity. The Staphylococcus epidermidis strain L within the variable regions.

mine whether the light or the heavy chain is in- Bacteria, cultured at 37 o c in T&-J-Hewitt broth for volved in the Fab-mediated binding of immuno- 16-18 h, were deposited by centrifugation at 1800 g for globulins to staphylococcal protein A. Expen- 15 min. Bacteria were washed three times in phosphate-

69

MATERIALS AND METHODS

The present investigation was designed to deter- 603, lacking protein A, was used as a negative control.

buffered saline, pH 7.2, containing 0.02% sodium azide and 0.05% Tween 20 (PBSA-Tween) and the bacterial concentration adjusted to lo9 organisms/ml, using o p tical density at 620 nm. For binding studies at different pH, bacteria were washed twice in PBSA-Tween and once in the corresponding buffer.

Immunoglobulins. Freeze-dried purified human poly- clonal IgG was purchased from AB Kabi, Stockholm, Sweden. The IgA F(ab')2 fragment of an IgA myeloma protein (HA, Kappa) with known alternative staphy- lococcal non-immune reactivity ( 8 ) was kindly provided by Professor S. G. 0. Johansson, Karolinska sjukhuset, Stockholm, Sweden. Five human IgA myeloma proteins (ROM, CAR, OLS, OVE, JOH) were isolated as de- scribed previously ( 1 8). Briefly, the IgA myeloma pro- teins were isolated by zone electrophoresis in agarose gel ( I I ) , followed by gel filtration either on a Sephadex G-200 column or on a Sephacryl S-300 column. IgG contaminating the IgA preparations was removed on an immunosorbent prepared by coupling rabbit anti-hu- man y-chain specific antibodies to Sepharose4B. Immu- noelectrophoresis of purified myeloma proteins ( 1 mp/ ml) showed a single precipitate when tested with antibo- dies to human serum proteins and to a- or y-chains. IgG and IgA were quantitated spectrophotometrically, using an extinction coefficient, E I",,, at 280 nm of 14.3 and 10.6, respectively (7).

Preparation of Fc and F(aby2 .fragments. Proteolytic fragments of polyclonal IgG were prepared and radio- labelled with I2'I as described earlier (4).

Preparation of light and heavy chains. Purified human polyclonal IgG was mildly reduced and alkylated as de- scribed earlier (5). SDS-PAGE analysis of isolated light and heavy chains under reduced and unreduced condi- tions showed no contamination of intact IgG. Isolated chains were 'zSI-labelled with lactoperoxidase in 0.1 M PBS buffer pH 6.0.

Quantitative binding experiments. IgG Fc and IgA binding assays were performed in duplicate in disposa- ble polystyrene tubes by mixing 25 pl of radiolabelled proteins with 2 x 10' bacteria suspended in 200 p1 PBSA-Tween buffer. After 30 min at room temperature, 2 ml of buffer was added, the bacteria deposited by centrifugation ( 1 800 g for 15 min) and the radioactivity of the pellet measured in a gamma counter. The amount of radiolabelled protein bound to the bacterial pellet was expressed as percentage of added radioactivity. Non-spe- cific binding was determined in parallel experiments, using the protein A-negative S. epidermidis strain L 603.

Isolated heavy chains may aggregate spontaneously at physiological pH levels ( 5 ) and the experimental condi- tions must be carefully determined when purified 7- chains are studied. Pilot experiments were therefore car- ried out with bacteria suspended in 0.1 M acetate (pH 3.5, 4.0, 4.5, 5.0 and 5 . 5 ) and in 0.1 M phosphate (pH 6.0, 6.5, 7.0, 7.5 and 8.0) buffers containing 0.02Yo so- dium azide and 0.05% Tween 20. Non-specific binding of both radiolabelled IgA ROM and polyclonal IgG Fc fragments to the negative control strain S. epidermidis L 603 was observed at low pH levels. Subsequent studies conducted with isolated chains were therefore performed

at pH 6.0, at which these proteins were stable and did not show non-specific binding.

Experiments using protein A-Sepharose CL4B, (lot no. IE 3202 1, Pharmacia Fine Chemicals, Uppsala, Swe- den), were performed with 100 p1 of a I % suspension as determined by the haematocrit. The binding was al- lowed to proceed at room temperature for 4 hours by slowly rotating the suspension. The Sepharose was pel- leted by centrifugation, half of the supernatant collected and radioactivity determined. From this measurement the radioactivity associated with the pellet was calcu- lated. Plain Sepharose CL-4B was used as a negative control.

Inhibition experiments. Three different types of inhi- bition experiments were performed (i) in the first expe- rimental set-up radiolabelled IgA F(ab')2 fragments were mixed with fixed amounts (5.9 and 59 pmol in 100 pI buffer) of intact IgA myeloma proteins and the binding of labelled material to protein A-Sepharose was mea- sured.

(ii) The capacity of intact polyclonal IgG, IgG F(ab')2- and Fc-fragments and monoclonal IgA OLS to inhibit the uptake of labelled IgA myeloma protein ROM to 2 x lo8 S. aureus organisms was determined with equimolar amounts (I25 - 235 pmol) of the inhibitors.

(iii) Increasing amounts (60 - 1920 pmol) of isolated, polyclonal immunoglobulin chains and intact IgG were mixed with 25 pI of the radiolabelled IgA myeloma protein ROM or polyclonal IgG Fc. The uptake to 5 x 10' S. aureus organisms was determined in 175 pI of 0. I M PBSA-Tween buffer, pH 6.0.

RESULTS

Protein A Reactive IgA Monoclonals The initial experiments were designed to select

IgA myeloma proteins expressing alternative pro- tein A reactivity, using the IgA F(ab')2 fragment of the myeloma protein HA as a reference. Five radiolabelled IgA myeloma proteins were tested

TABLE I . Capacity of Equimolar Amounts (125 - 235 pmol) of Purified Immunoglobulin Preparations to Inhi- bit the Binding of 0.05 pg.Radiolabelled IgA Myeloma Protein ROM to 2 x lon S. aureus Organisms (Strain

Cowan I)

Inhibitor Inhibiting capacity' %

Polyclonal IgG 82 Polyclonal IgG F(ab')2 35 Polyclonal IgG Fc 0 IgA myeloma protein OLS 90

a Inhibition expressed as percent reduction in binding of radiolabelled protein.

70

- 140 -

- - 120 - I: D a 100 - Y

< P - 80 - r 0

,“ 60 -

a 3 40 - ap -

20 -

6 c

- 0 - r 1 I

140

0

2 100 C

D

L cha ins

- 80

Y - a 4 0 1

2

ap 20

1 O J , I I I I I

80 120 240 480 960 1020 60 120 240 480 060 1920

amount o f p r o t e i n a d d e d ( p m o l ) amount o f p r o t e i n a d d e d (pmol )

Fig. 1. Capacity of intact polyclonal IgG (IgG) and isolated light (L) and heavy (H) chains to inhibit the binding of 0.05 pg radiolabelled IgA myeloma protein ROM (A) and 0.01 pg polyclonal IgG Fc (B) at pH 6.0 to the S. aureus strain Cowan 1. The uptake is expressed as percentage of the binding measured without inhibitor. Error bars denote arithmetic mean +/- half difference of values of duplicate determinations.

for quantitative binding to the S. aureus Cowan I strain. Three myeloma proteins (ROM, OLS, OVE) showed positive binding. Myeloma protein ROM showed the highest uptake level, 4396, and was used in subsequent experiments.

Inhibition experiments were performed to con- firm that the protein A reactivity seen among intact IgA myeloma proteins was restricted to the F(ab’)2-portion and independent of the Fc-part of the molecule. The three IgA myeloma proteins which showed reactivity in direct binding experi- ments inhibited the uptake of radiolabelled IgA F(ab’)2 fragments to protein A-Sepharose. In con- trast, the two non-reactive myeloma proteins were non-inhibitory. Although these results strongly suggest that the binding of intact IgA molecules is mediated exclusively by structures in the F(ab’)2- portion, further experiments were conducted to substantiate this observation. The binding of IgA ROM was as effectively inhibited by polyclonal IgG as by intact IgA molecules (Table 1). A de- finite but less marked inhibition was achieved by IgG F(ab’)2 fragments. IgG Fc fragments were completely non-reactive. A similar pattern was observed when protein A-Sepharose was used ins- tead of staphylococci. Inhibition by Immunoglobulin Chains

Because the IgA myeloma protein ROM did not express Fc binding properties, the intact IgA mole- cule could be used to assess the F(ab’f2-mediated

interaction with immunoglobulin chains. Isolated light and heavy immunoglobulin G chains pre- pared from polyclonal IgG were used in inhibition experiments to localize the binding site within the F(ab’)2 fragment. Binding of IgA ROM was inhi- bited dose-dependently by intact polyclonal IgG and by isolated heavy chains (Fig. 1A). Isolated light chains were non-inhibitory. Half-maximal inhibition was noted with 300 pmol IgG. The y-chain preparation was less effective than the intact molecule, requiring 1200 pmol to reach the same degree of inhibition. At 1920 pmol, intact IgG and heavy chains inhibited 97% and 69%, respectively, of the IgA ROM binding to S. aureus organisms.

Analogous experiments were conducted with radiolabelled IgG Fc fragments. A dose-dependent inhibition was noted with intact IgG and y-chains but not with light immunoglobulin chains (Fig. 1B). Half-maximal inhibition was noted at 270 pmol of IgG and at 730 pmol of the heavy y-chain preparation. At 1920 pmol, intact IgG and heavy chains inhibited 85% and 8896, respectively, of the IgG Fc binding.

Purified light and heavy chains as well as intact IgG were radiolabelled and tested for binding to protein A-Sepharose. An uptake of 58% was recorded with y-chains, while the light chain prep- aration showed only a 7% binding. A 75% binding was seen with intact IgG molecules.

71

DISCUSSION Protein A can interact in two non-immune ways with immunoglobulins (6, 8). The classical Fc-me- diated binding has been demonstrated in three out of four human IgG subclasses and in IgG from several mammalian species (1 5, 16). A two-step reaction was postulated for the precipitation of protein A by human IgG ( I 3). The primary step included IgG Fc binding to protein A. Evidence for a secondary step independent of the Fc portion was presented. An interaction with structures in the variable portion of the light and heavy chains, i.e. the Fab parts, was suggested. Zngands de- scribed the F(ab’)Zmediated non-immune inter- action of immunoglobulins with protein A (8, 10). This alternative, non-immune interaction was not only restricted to IgG but was described in the immunoglobulin classes A, M and E as well. Zn- gands & Johansson (9) showed that both the IgG Fc- and F(ab’)Zmediated non-immune inter- actions were needed in the protein A precipitation reaction.

The present investigation confirms earlier re- ports (1, 8) and shows that the interaction between staphylococcal protein A and human IgA is medi- ated by structures exposed on the F(ab’)2 portion of the molecule. In our experiments we further demonstrated that the interaction with the Fab portion is localized on the heavy immunoglobulin chain. Inganas has compared the inhibiting capac- ity of intact IgA and IgA F(ab’)2 fragments wi- thout noting any significant difference in potency (8). Because there is no interference through Fc- mediated mechanisms and no difference in bind- ing properties, intact IgA molecules can be used for studies of the alternative F(ab’)2-mediated pro- tein A reactivity. The binding pattern observed with intact S. aureus organisms and protein A-Se- pharose was identical, although higher binding levels were generally achieved with bacterial cells.

The F(ab’)2-reactive structure is not restricted to IgA but can also be demonstrated in the other immunoglobulin classes including IgG (3, 8, 10). Immunoglobulin chains prepared from polyclonal IgG could therefore be used to determine the type of chain involved in the alternative protein A binding. Our observation that the heavy, but not the light, chain preparation inhibited the IgA binding shows that the reactive site is part of the y-chain. On a molar basis, intact IgG was a more potent inhibitor than isolated heavy chains. Al- though the purified light chains per se is non-reac- tive it may passively stabilize the intact immuno- globulin molecule and thereby enhance the binding capacity of the intact molecule. The dif-

ference in binding potency can also be explained by the monovalency of isolated heavy chains or by conformational changes known to occur on reduc- tion of the IgG molecule (2). Other investigators have previously presented indirect evidence for heavy-chain involvement. For example, Ingands did not find any correlation between the type of light chain and F(ab’f2-mediated reactivity (8). Similar observations were made by Biguzzi, who also noted that the purified light chain was non- reactive (1).

The y-chain portion of the Fab fragment is composed of a variable domain (VH) and a cons- tant domain (CHI). The present study does not give any information on which of the domains within the Fab fragment cames the protein A binding site. Biguzzi performed experiments with IgM Fv fragments, which are composed of the variable heavy (VH) and light (VL) domains. Fv fragments from a subset of immunoglobulins showed protein A reactivity. Light chains were non-reactive and the kappa/lambda ratio was equal in reactive and non-reactive myeloma pro- teins. He suggested therefore a heavy chain local- ization of the Fab-mediated protein A interaction (I) . The results of Biguzzi and our study point to the variable heavy domain, VH, as the binding site.

In our study the binding of IgA to protein A-carrying organisms was inhibited by IgG and F(ab’)2 fragments but not by Fc fragments. Simi- lar results have been reported by Inganus, who found that the Fc and IgE binding to protein A-Sepharose did not interfere, suggesting that the two reactivities are independent of each other (8, 10). The present inhibition experiments were per- formed with a ninefold excess of Fc fragments over the number of protein A-binding sites calcu- lated by Kronvall et al. (14). The affinity of Fc fragments for protein A is 10 - 15 times higher than that of reactive F(ab’)2 fragments (1). The observation that no inhibition was achieved with purified Fc fragments implies that the Fc fragment did not compete with the F(ab’)2 fragment for common binding sites on the protein A molecule. It is therefore reasonable to suggest that there are two separate binding sites, one for each of the two types of fragments. Even if the two sites do not physically overlap, they could still have a struc- tural relationship. Considerable homology in amino acid sequence between constant domains and the framework part of the variable domain has in fact been documented (1 7). This homology could be a common basic structure expressing non-immune immunoglobulin reactivity.

72

Recent data have shown similarities between non-immune immunoglobulin interactions of protein A-carrying S. aureus and of group C and G streptococci. These bacterial species possess sur- face components, IgG receptors, with affinity for both the Fc and the Fab portion of IgG (4, 10, 12, 19, 20). The Fab interaction of group C and G streptococci is also mediated by structures on the IgG heavy chain (5). Though the staphylococcal and the streptococcal non-immune interactions with Fab structures are analogous, differences ex- ist in the specificity for immunoglobulin classes and for different mammalian immunoglobulin (4, 8, 10).

The excellent technical assistance of Mrs Elisabeth Nilsson is gratefully acknowledged. This investigation was supported by grants from ‘Forenade Liv’ Mutual Group Life Insurance Company, Stockholm, Sweden, the Swedish Society of Medical Sciences, the A . Osterlund Foundation, and the Royal Physiographical Society in Lund.

REFERENCES

I . Biguzzi, S.: Fcy-like determinants on immunoglo- bulin variable regions: identification by staphy- lococcal protein A. Scand. J. Immunol. 15: 605- 618, 1982.

2. Dorrington, K. J. & Smith, B. R.: Conformational changes accompanying the dissociation and associ- ation of immunoglobulin-G subunits. Biochim. Biophys. Acta 263: 70-81, 1972.

3. Endresen, C.: The binding to protein A of immuno- globulin G and of Fab and Fc fragments. Acta path. microbiol. a n d . Sect. C, 87: 185- 189, 1979.

4. Emtell, M., Myhre, E. B. & Kronvall, G.: Alter- native non-immune F(ab’)Z-mediated immunoglo- bulin binding to group C and G streptococci. Scand. J. Immunol. 17: 201-209, 1983.

5. Ernlell. M., Myhre, E. B. & Kronvall. G.: Non-im- mune IgG F(ab’)2 binding to group C and G strep tococci is mediated by structures on y chains. Scand. J. Immunol. 21: 151-157, 1985.

6 . Forsgren, A . & Sjoquist, J.: Protein A from S. aureus. 1. Pseudoimmune reaction with human y- globulin. J. Immunol. 97: 822-827, 1966.

7. Hudson, L. & Hey, F. C.: Practical Immunology. 2nd ed. Blackwell Scientific Publications, Oxford

8. Inganas, M.: Comparison of mechanisms of inter- action between protein A from Staphylococcus

1980, pp. 346-349.

9.

10.

I I .

12.

13.

14.

15.

16.

17.

18.

19.

20.

aureus and human monoclonal IgG, IgA and IgM in relation to the classical F q and the alternative F(ab’)2 E protein A interactions. Scand. J. Immu- nol. 13: 343-352, I98 I . Inganas, M. & Johansson, S. G. 0.: Influence of the alternative protein A interaction on the precipita- tion between human monoclonal immunoglobulins and protein A from Staphylococcus aureus. Int. Archs Allergy appl. Immun. 65: 91-101, 1981. Inganas, M.. Johansson, S. G. 0. & Bennich, H. H.: Interaction of human polyclonal IgE and IgG from different species with protein A from Staphy- lococcus aureus: Demonstration of protein-A-reac- tive sites located in the F(ab’)2 fragment of human IgG. Scand. J. Immunol. 12: 23-31, 1980. Johansson, B. G.: Agarose gel electrophoresis. Scand. J. clin. Lab. Invest. 29: Suppl. 124, 7-19, 1972. Kronvall, G.: A surface component in group A, C and G streptococci with non-immune reactivity for immunoglobulin G. J. Immunol. 111: 1401-1406, 1973. Kronvall, G., Messner, R. P. & Williams, JR., R. C.: Immunochemical studies on the interaction be- tween staphylococcal protein A and @ globulin. J. Immunol. 105: 1353-1359, 1970. Kronvall, G.. Quie, P. G. & Williams, R. C.: Quant- itation of staphylococcal protein A: determination of equilibrium constant and number of protein A residues on bacteria. J. Immunol. 104: 273-278, 1970. Kronvall, G.. Seal, U. S.. Finstad, J. & Williams. JR., R. C.: Phylogenetic insight into evolution of mammalian Fc fragment of rC; globulin using staphylococcal protein A. J. Immunol. 104: 140- 147, 1970. Kronvall, G. & Williams, JR., R. C.: Differences in anti-protein A activity among IgG subgroups, J. Immunol. 103: 828-833, 1969. Marquart. M. & Deisenhofer, J.: The threedimen- sional structure of antibodies. Immunol. Today 3:

Myhre, E. B. & Erntell, M.: A non-immune inter- action between the light chain of human immuno- globulin and a surface component of a Peptococcus magnus strain. Molec. Immunol. 22: 879-885, 1985. Myhre, E. B. & Kronvall, G.: Heterogeneity of non- immune immunoglobulin Fc reactivity among gram-positive cocci: description of three major types of receptors for human immunoglobulin G. Infect. Immun. 17:475-482, 1977. Myhre, E. B. & Kronvall, G.: Immunochemical aspects of Fc-mediated binding of human IgG sub classes to group A, C and G streptococci. Molec. Immunol. 17: 1563-1573, 1980.

160- 166, 1982.

73