Immunology, 1975, 29, 921.
Fragmentation of Human IgG by a New Protease Isolated from the Basidiomycete Armillaria mellea I. M. HUNNEYBALL AND D. R. STANWORTH
Department of Experimental Pathology, University of Birmingham
(Received 11 th April 1975; acceptedfor publication 18th April 1975)
Summary. Digestion of human IgG by a new lysine-specific protease, isolated from the basidiomycete Armillaria mellea, produced Fc and Fab fragments similar to those produced by papain digestion of the same molecule. Digestion appeared to be restricted to a single cleavage point within the hinge region of the IgG molecule. Myeloma proteins of IgGl, IgG3 and IgG4 subclasses were found to be digested at an extremely rapid rate whereas IgG2 myeloma proteins appeared to be resistant to digestion by this enzyme. INTRODUCTION Proteolytic degradation of IgG into macromolecular fragments has been widely exploited in studies aimed at defining the location and structural basis of the various biological characteristics demonstrated by antibody molecules. For instance, such an approach has been adopted in the localization of 'auto-antigenic' sites against which are directed the anti-y-globulins found in the sera of patients with rheumatoid arthritis (Goodman, 1961; McDuffie, Oikawa and Nishi, 1965; Stewart, Smith and Stanworth, 1973). Thus, the Fc fragment, which constitutes approximately one third of the IgG molecule, has been shown to possess the binding site(s) for 'general' rheumatoid factors, i.e. those not directed against allotypic determinants (Stewart et al., 1973; Stewart, Hunneyball and Stanworth, 1975). In contrast, both of the fragments produced by plasmin cleavage of IgG (i.e. pFc' and Facb fragments) were found to be unreactive towards these rheumatoid factors. It was of some interest, therefore, to submit IgG to proteolysis by a new lysine-specific protease, recently isolated from the fruiting body of the basidiomycete Armillaria mellea, to ascertain whether it was possible to produce new types of cleavage fragment, particularly originating from the Fc region, which would permit the more precise localization of antigenic sites and other types of effector group. This A. mellea protease has previously been shown to be an endopeptidase which cleaves proteins at peptide bonds N-terminal to lysine residues and, in certain instances, C-terminal to arginine residues (Doonan et al., 1974; Walton, Turner and Broadbent; UK patent number 1263956, 1972).
MATERIALS AND METHODS Materials A. mellea protease was kindly supplied by Dr P. L. Walton (Pharmaceuticals Division, I.C.I. Ltd, Macclesfield). Sheep erythrocytes were obtained from Burroughs Wellcome Correspondence: Dr I. M. Hunneyball, Department of Experimental Pathology, University of Birmingham,
Birmingham BI5 2TJ.
921
I. M. Hunneyball and D. R. Stanworth Ltd, as sheep blood in Alsever's solution. Unless otherwise stated, all general laboratory reagents were obtained as 'Analar' grade from B.D.H. Ltd. Pooled human IgG was isolated from serum by precipitation with 33 per cent saturated ammonium sulphate and then purified by batch ion-exchange chromatography using diethylaminoethyl cellulose (Whatman DE52) (Stelos, 1967; Stanworth, 1960). Human IgGl, IgG2 and IgG3 myeloma proteins were prepared in an identical manner. Human IgG4 myeloma protein was isolated by ion-exchange chromatography on a column (90 x 2-2 cm) of DEAE-cellulose (Whatman DE52) equilibrated with 0-01 M phosphate buffer, pH 6-3, by elution with a sodium chloride gradient (0.001-0-05 M), IgG4 eluting at a position corresponding to 0-02 M sodium chloride. Purity of the IgG preparations was assessed by immunoelectrophoresis and analytical ultracentrifugation at a concentration of 10 mg/ml. In addition, the myeloma proteins were found to be pure by both immunodiffusion using subclass specific antisera and Gm typing. The Fc and Fab fragments of human IgG were prepared by digestion of IgG with papain (Sigma Chemical Company, Ltd) in the presence of cysteine and purified by gel-filtration on Sephadex G-150 (Pharmacia Ltd), followed by ion-exchange chromatography on carboxymethyl Sephadex (Sephadex C50, Pharmacia Ltd) and diethylaminoethyl cellulose (Whatman DE52) according to Franklin and Prelli (1960).
922
Antisera Sheep antisera to human IgG, human Faby fragment, and the y chain were kindly provided by Dr D. Catty (Department of Experimental Pathology, University of Birmingham). Antiserum to the Fc fragment was prepared by absorption of the sheep antihuman y chain antiserum with pure Fab fragment. Specific anti-Cy2 domain antiserum was obtained by absorbing sheep anti-Fc antiserum with pFc' fragment (Cy3) domain, prepared according to Turner and Bennich (1968), which had been insolubilized using ethyl chloroformate according to Avrameas and Ternynck (1967). Antiserum to the Cy3 domain was prepared by injecting guinea-pigs with pFc' fragment in Freund's complete adjuvant. Baboon antiserum to sheep erythrocytes was kindly supplied by Mr G. A. Stewart (Department of Experimental Pathology, University of Birmingham). Rheumatoid arthritis sera were obtained from the Queen Elizabeth Hospital (Birmingham) by courtesy of Dr C. F. Hawkins.
Immunoelectrophoresis and immunodifusion Immunoelectrophoresis was performed in a Shandon electrophoresis apparatus using 1 per cent Oxoid Agar number 3 in barbitone buffer, pH 8-6 (I = 0.05). Electrophoresis was carried out at a constant current of 18 mA per plate for 1-5 hours. Immunodiffusion was allowed to proceed for 24 hours at 4V.
N-terminal analysis N-terminal analysis was performed according to the method of Gray (1972) employing dansyl chloride. Reactivity with 'general' rheumatoidfactors The reactivity of human IgG subfragments with 'general' rheumatoid factors was measured by a haemagglutination-inhibition technique employing diluted rheumatoid
923 Fragmentation of Human IgG92 serum and sheep erythrocytes sensitized with a subagglutinating dose of baboon anti-sheep erythrocyte antiserum according to Stewart et al. (1975). Prior to testing, the rheumatoid sera were clarified by centrifugation, decomplemented by incubation at 560 for 20 minutes and absorbed with an equal volume of packed sheep erythrocytes. The rheumatoid serum was diluted with 0*0l m phosphate-buffered saline (0.15 M), pH 7-2, to give an agglutination titre of 1 in 4 with sensitized erythrocytes. To serial doubling dilutions (0-025 ml) of inhibitor (at an initial concentration of 10 mg/ml) in the wells of a microtitre tray was added an equal volume of the diluted rheumatoid serum. After incubation for 1 hour at 370, an aliquot (0-025 ml) of a 1 per cent suspension of sensitized cells was added to each well. The inhibition titres were read after incubation for a further hour.
Gm typing The expression of allotypic markers on the subfragments was determined by a haemagglutination-inhibition technique employing human 0 Rh'+ erythrocytes sensitized with incomplete anti-D antibodies of known Gm type and sera from normal transfused blood donors. RESULTS DIGESTION CONDITIONS
Pooled normal human JgG (20 mg/ml) in 0-0l m Tris-HC1 buffer, pH 7-0, containing 0 -l15 m sodium chloride and 0*002 m calcium acetate, was digested with A. mellea protease at an enzyme: substrate ratio of 1 :1I00 (wt/wt) at 3 7'. Serial samples were removed at intervals between 10 seconds and 24 hours and the enzyme inactivated by addition of an equal volume of 0-0l m disodium. EDTA in 0-01 m phosphate-buffered saline (0-l5 m), pH 7-2. Immunoelectrophoresis, using sheep anti-IgG antiserum, indicated that digestion had occurred almost instantaneously and that no further increase in digestion was evident even after 24 hours. This finding was substantiated by gel filtration analysis on a column (90 x 2-2 cm) of Sephadex G-150 equilibrated with 0'05 m ammonium carbonate buffer, pH 8 -6. The optimum digestion time was found to be 3 hours using an enzyme : substrate ratio of 1:1I000 (wt/wt). ISOLATION OF FRAGMENTS
The digestion products were separated on a column (90 x 3 -2 cm) of Sephadex G- 150 equilibrated with 0-05 m ammonium carbonate buffer, pH 8-6. Three major protein fractions could be distinguished from the elution profile (Fig. 1): fraction 1 eluted in a position corresponding to undigested IgG; fraction 2 eluted in a position similar to papain Fc and Fab fragments; fraction 3 contained dialysable material. Fractions 1 and 2 were purified by rechromatography on a column (90 x 2 -2 cm) of Sephadex G- 150 equilibrated with 0-05 m ammonium carbonate buffer, pH 8-6, and characterized by immunoelectrophoresis against a variety of antisera (Fig. 2a). Fraction 1 consisted of a single component, precipitating with both anti-Fc and anti-Fab antisera, with an electrophoretic mobility identical to that of native human IgG. Fraction 2 contained two components, exhibiting non-identity against sheep anti-JgG antiserum, with electrophoretic mobilities similar to those of the papain Fc and Fab fragments. The Fc-like fragment precipitated with the I
924
L M. Hunneyball and D. R. Stanworth Or-
/I' I)
E
\
I
0
0-5 0
0
X,1 I \ L
o00
L
200 300 Elution volume (ml)
\
3
400
FIG. 1. Gel filtration of a 3-hour A. mellea protease digest of pooled human IgG on a column (90 x 3-2 cm) of Sephadex G-150 equilibrated with 0 05 ammonium carbonate buffer, pH 8-6.
FIG. 2 (a) Immunoelectrophoresis of Sephadex G-150 fractions 1 and 2 of a 3-hour A. mellea protease digest of human IgG, using sheep anti-IgG (A), sheep anti-Fc (B), and sheep anti-Fab (C) antiserum. Native IgG (N) was included as a control. (b) Immunoelectrophoresis of: (1) papain Fc fragment; (2) A. mellea protease fraction 2(2); (3) A. mellea protease fraction 2(1); (4) papain Fab fragment (using sheep anti-IgG antiserum). (c) Gel diffusion analysis of: (1) papain Fc fragment; (2) A. mellea protease fraction 2(2); (3) A. mellea protease fraction 2(1); (4) papain Fab fragment (using sheep anti-IgG antiserum (A). Well (5) contained buffer only.
anti-Fc antiserum but not the anti-Fab antiserum; whereas the Fab-like fragment precipitated with the anti-Fab antiserum but not the anti-Fc antiserum (Fig. 2a.) Human IgG1 myeloma proteins were found to behave in an identical manner to pooled human IgG on treatment with A. mellea protease. However, unless otherwise stated, normal pooled human IgG was used. Due to the similarity between the fraction 2 components and papain Fc and Fab fragments, separation of the fraction 2 components was achieved by the procedure previously adopted for the separation of the papain fragments, i.e. ion-exchange chromatography on carboxymethyl Sephadex and diethylaminoethyl cellulose by the method of Franklin and Prelli (1960) as outlined in Fig. 3. Fraction 2 was dialysed exhaustively
925 Fragmentation of Human IgG against 0*01 M phosphate buffer, pH 7-6, and applied to a column (60 x 2-2 cm) of CM Sephadex (C50, Pharmacia Ltd) equilibrated with the same buffer. As will be seen from Fig. 4a, two fractions were resolved by stepwise elution with 0-01 M phosphate buffer, pH 7-6 (fraction 2A) and 0 3 M phosphate buffer, pH 7-6 (fraction 2B). Fraction 2A was concentrated, dialysed against 0-01 M phosphate buffer, pH 8-0, and applied to a column (60 x 2-2 cm) of DEAE-cellulose (Whatman DE-52) equilibrated with the same buffer. As will be seen from Fig. 4b, two fractions were resolved by stepwise elution with 0.01 M phosphate buffer, pH 8-0 (fraction 2A(1)) and 0-3 M phosphate buffer, pH 8-0 (fraction 2A(2)). Fraction 2B was purified in a identical manner giving rise to two fractions: 2B(1) and 2B(2) as shown in Fig. 4c. Fractions 2A(1) and 2B(1) were pooled and designated 2(1). Similarly, fractions 2A(2) and 2B(2) were pooled and designated 2(2). Human IgG Am protease, pH 7 0
(1:1000enzyme: substrate) 3 hours at 370C Sephadex G-150 3
2
(Peptides)
(IgG) CM Sephadex pH 76
03 M
|O.I M
2B DEAE-cellulose pH 8-0
2A DEAE-cellulose pH 8-0
00I M
01 3M
2A(1)
2A(2)
2(1)
OCOI M 2B(l)
0 3M
2B(2)
2(2)
FIG. 3. Outline of purification scheme for A. mellea protease subfragments of pooled human IgG.
Both fractions appeared pure by immunoelectrophoresis at a concentration of 5 mg/ml using sheep anti-IgG antiserum (Fig. 2b). However, slight contamination of fraction 2(1) with fraction 2 (2), and vice versa, was evident on gel diffusion analysis of the two fractions at 5 mg/ml against the same antiserum (Fig. 2c). CHARACTERIZATION OF THE FRAGMENTS
On immunoelectrophoresis (Fig. 2b) fraction 2(1) demonstrated similar mobility to the papain Fab fragment. In contrast, fraction 2(2) was found to be slower electrophoretically than the papain Fc fragment. The results of gel diffusion analyses of the purified fractions are summarized in Table 1. Fraction 2 (1) gave a reaction of complete identity with a preparation of the papain Fab fragment on gel diffusion against anti-IgG (Fig. 2c) and anti-Fab antisera. Fraction 2 (2) gave a reaction of complete identity with a preparation of the papain Fc fragment on gel diffusion against anti-IgG, anti-Fc, anti-Cy2 domain and
I. M. Hunneyball and D. R. Stanworth
926
(a) I'
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40
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Elution volume (ml)
FIG. 4. (a) Purification of Sephadex G150 fraction 2 on a column (60x2 2 cm) of carboxymethyl Sephadex (C50) equilibrated with 0-01 M phosphate buffer, pH 7-6. Fraction 2A was eluted with 0-01 M phosphate buffer, pH 7-6. Fraction 2B was eluted with 0-3 M phosphate buffer, pH 7-6. (b) Purification of CM Sephadex fraction 2A on a column (60 x 2-2 cm) of DEAE-cellulose (Whatman DE52) equilibrated with 0-01 M phosphate buffer, pH 8-0. Fraction 2A(1) was eluted with 0-01 M phosphate buffer, pH 8f0. Fraction 2A(2) was eluted with 0 3 M phosphate buffer, pH 8-0. (c) Purification of CM Sephadex fraction 2B on a column (60 x 2-2 cm) of DEAE-cellulose (Whatman DE52) equilibrated with with 0-01 M phosphate buffer, pH 8-0. Fraction 2B(l) was eluted with 0-01 M phosphate buffer, pH 8-0. Fraction 2B(2) was eluted with 0 3 M phosphate buffer, pH 8-0. TABLE 1 GEL-DIFFUSION PRECIPITIN ANALYSIS OF PURIFIED PREPARATIONS OF A. mellea PROTEASE DIGESTION FRAGMENTS OF POOLED HUMAN IgG (FRACTIONS
2(1)
AND
2(2)) USING VARIOUS SPECIFIC ANTISERA
Antiserum
Fraction 2 (1)
Fraction 2 (2)
Sheep anti-IgG Sheep anti-Fab Sheep anti-Fc Sheep anti-Cy2 Guinea-pig anti-Cy3
+ + -
+ + + +
927 Fragmentation of Human IgG anti-Cy3 domain antisera. Analytical ultracentrifugation indicated that both fractions sedimented at a similar rate to the papain Fc fragment (Table 2). N-terminal analysis of fraction 2(2) which had been prepared from an IgG1 myeloma protein, indicated the presence of an N-terminal lysine residue. This contrasted with the presence of a threonine residue at the N-terminus of the papain Fc fragment of the same myeloma protein. TABLE 2 SEDIMENTATION COEFFICIENTS OF PURIFIED A. mellea PROTEASE DIGESTION FRAGMENTS OF POOLED HUMAN IgG (FRACTIONS 2(1) AND 2(2)) AT A CONCENTRATION OF 4 mg/ml IN 0-01 M PHOSPHATE-BUFFERED SALINE (0.15 M), pH 7-2
Sample
S20,w
Pooled human IgG Papain Fc fragment Fraction 2(1) Fraction 2 (2)
6-82 + 0 05 3-92 + 0)04 3-74+0 06 3 85+ 0 06
SUBFRAGMENTATION OF THE Fc FRAGMENT
Subfragmentation of papain Fc fragment by A. mellea protease was attempted. Pooled human IgG Fc fragment (20 mg/ml) in 0-01 M Tris-HCl buffer, pH 7 0, containing 0-15 M sodium chloride and 0-002 M calcium acetate, was digested with A. mellea protease at an enzyme:substrate ratio of 1:100 (wt/wt) at 370. Serial samples were removed at intervals between 10 seconds and 48 hours and the enzyme inactivated by addition of an equal volume of 0-01 M disodium EDTA in 0 01 M phosphate-buffered saline (0.05 M), pH 7-2. The samples were analysed by immunoelectrophoresis, using sheep anti-Fc antiserum. Evidence of some proteolysis was apparent after 6 hours, but even after 24 and 48 hours digestion the extent of cleavage indicated by immunoelectrophoretic analysis was very limited. This conclusion was confirmed by analytical gel filtration of the 24- and 48-hour digests on a column (90 x 2-2 cm) of Sephadex G-100 equilibrated with 0 05 M ammonium carbonate buffer, pH 8-6 (Fig. 5). Thus it would appear that under the conditions em0*75
E
-
0 50 -l
i
co'1
0
O 025
II
0
100
I\
. ' 200
300
400
Elution volume (ml)
FIG. 5. Gel filtration of a 48-hour A. mellea protease digest of human IgG Fc fragment on a column (90 x 2-2 cm) of Sephadex G-100 equilibrated with 0 05 M ammonium carbonate buffer, pH 8-6.
I. M. Hunneyball and D. R. Stanworth 928 ployed for fragmentation of IgG, the papain Fc fragment is essentially resistant to digestion by A. mellea protease.
DIGESTION OF IgG SUBCLASSES
Preliminary investigations into the susceptibility of the four subclasses of human IgG by A. mellea protease were performed. IgG myeloma proteins (10 mg/ml) in 0 01 M phosphate buffer, pH 7-0, containing 0*15 M sodium chloride and 0-002 M calcium acetate were digested at an enzyme:substrate ratio of 1:100 (wt/wt) at 370 for 30 minutes. Excess solid disodium EDTA was added to terminate digestion and the digests analysed by immunoelectrophoresis (Fig. 6). Control myeloma proteins were treated in an identical manner but in the absence of enzyme. IgG1 and IgG3 myeloma proteins seemed to be completely digested by the A. mellea protease, whereas IgG4 appeared to be only partially digested. In contrast, IgG2 myeloma protein seemed to be resistant to digestion.
FIG. 6. Immunoelectrophoresis of A. mellea protease digests of myeloma proteins of the four subclasses of human IgG, using sheep anti-IgG antiserum. Number denotes subclass of the myeloma protein. AUTO-ANTIGENICITY OF THE FRAGMENTS
The capacity of the purified subfragments of pooled IgG to react with 'general' rheumatoid factors (i.e. those not directed against allotypic determinants) was investigated, the results of which are summarized in Table 3. Fraction 2 (1) showed no reactivity towards TABLE 3 CAPACITY OF A. mellea PROTEASE DIGESTION FRAGMENTS OF POOLED HUMAN IgG TO INHIBIT THE AGGLUTINATION, BY RHEUMATOID ARTHRITIS SERUM, OF SHEEP ERYTHROCYTES SENSITIZED WITH BABOON IgG. (THE RHEUMATOID ARTHRITIS SERUM WAS DILUTED TO GIVE A TITRE OF 1 IN 4 WITH SHEEP ERYTHROCYTES SENSITIZED WITH BABOON IgG)
Sample IgG Fraction 2 (1) Fraction 2 (2) Papain Fc Papain Fab
Lowest concentration (mg/ml) of protein or fragment giving inhibition
0-6 >5 0
03 03 >5 0
929 Fragmentation of Human IgG 'general' rheumatoid factors whereas fraction 2(2) reacted to the same extent as the papain Fc fragment. The localization of Gm and InV allotypic markers on the purified subfragments of pooled IgG was also investigated; fraction 2(1) expressed the Gm (f) and InV (1) markers, whereas fraction 2(2) expressed Gm (a), (x), (b1) and (b4) markers. DISCUSSION ofhuman mellea Cleavage IgG byA. protease was found to proceed at a muchfaster rate than that by other common proteolytic enzymes such as papain and pepsin. Similar kinetics have been demonstrated for the action of this enzyme on casein and fibrinogen (Walton, personal communication); moreover, the action of A. mellae protease on these proteins produced large fragments relatively resistant to further digestion by the enzyme, a feature exhibited by the enzyme in its action on native IgG and substantiated by the resistance of the papain Fc fragment to digestion by the enzyme. The products ofA. mellea protease digestion of human IgG appear to be similar immunochemically to the Fc and Fab fragments produced by papain digestion of the same molecule. The Fab-like fragment produced by A. mellea protease digestion (fraction 2(1)) proved to be identical to the papain Fab fragment by all criteria applied. The Fc-like fragment produced by A. mellea protease digestion (fraction 2(2)) was identical to the papain Fc fragment by gel diffusion analysis with specific heteroantisera, analytical'ultracentrifugation, and its capacity to react with 'general' rheumatoid factors; but it was electrophoretically slower than the papain Fc fragment and contained lysine at the N-terminus in contrast to the N-terminal threonine of the papain Fc fragment. The slower electrophoretic mobility of fraction 2(2) indicates a higher content of basic residues which would be consistent with cleavage of IgGl by A. mellea protease at the Asp-Lys bond at position 221-222 within the hinge region of the molecule (Fig. 7) (assuming the sequence of the IgGi myeloma protein Eu described by Edelman, Cunningham, Gall, Gottlieb, Rutishauser and Waxdal, 1969). Thus fraction 2(2) would differ from the papain Fc fragment by only three amino acid residues at the N-terminus of the fragment. This proposed similarity between fraction 2(2) and the papain Fc fragment is supported by the resistance of both fragments to subsequent digestion by A. mellea protease. Am protease
N
L
)
Papain
II
S N
S
N
sA
by
S~~ ~
SI
Y
C
~~~ I r
I~~~~
s
C
N
FIG. 7. Probable location of the point of cleavage of IgG by A. mellea protease. Residue numbering is based on the sequence of the IgGl myeloma protein Eu (Edelman et al., 1969).
930 I. M. Hunneyball and D. R. Stanworth Cleavage by A. mellea protease at the hinge region of IgG, is consistent with the observed expression of allotypic markers Gm (a), (x), (b') and (b4) on the Fc-like fragment and of Gm (f) and InV(l) on the Fab-like fragment. Cleavage of peptide bonds N-terminal to lysine residues is in accord with the reported primary specificity of A. mellea protease for peptide bonds within other proteins, such as casein, fibrinogen and aspartate aminotransferase (Doonan et al., 1974; Walton, personal communication). Therefore, the cleavage of IgG by A. mellea protease appears to be similar in all respects to the reported mode of action of the enzyme on other protein molecules. The limited specificity of A. mellea protease with regard to cleavage of human IgG appears to parallel the restricted specificity of plasmin for the hinge region of the molecule; the difference in specificity between the two enzymes being reflected by the position of the bond cleaved relative to the lysine residue, i.e. plasmin cleaves peptide bonds C-terminal to lysine, whereas A. mellea protease cleaves peptide bonds N-terminal to lysine residues. A. mellea protease appeared to cleave IgG3 and IgG4 in a similar fashion to IgGl, producing two subfragments. The susceptibility of the different subclasses to cleavage by the enzyme was found to be similar to the reported susceptibility of the proteins to digestion by papain (Gergely, Fudenberg and Van Loghem, 1970). In contrast to papain, however, the difference in susceptibility to digestion by A. mellea protease between the subclasses was found to be more definitive, i.e. IgGl and IgG3 appeared to be digested completely to Fc and Fab fragments, whereas no trace of digestion of IgG2 was evident under the same conditions. This finding indicates the potency of this enzyme for subclass determination of myeloma proteins. A. mellea protease appears to be an enzyme of great potential for producing large subfragments from protein molecules for studies of the submolecular localization of various biologically active sites. With regard to cleavage of human IgG, this protease can rapidly produce Fc and Fab fragments in high yield, due to the resistance of these subfragments to further degradation by the enzyme. The increased size of the A. mellea protease Fc fragment in comparison with that produced by papain cleavage may be of considerable use in the localization of hinge region-located effector sites. However, from this study, it was not possible to gain any further information with regard to the localization of the 'general' rheumatoid factor-reactive determinants within the Fc fragment. ACKNOWLEDGMENTS The Gm typing of the myeloma proteins and fragments was kindly performed by Miss D. F. Barr (Regional Blood Transfusion Service). Generous financial support from the Medical Research Council is gratefully acknowledged. REFERENCES
AVRAMEAS, S. and TERNYNCK, T. (1967). 'Biologically active water-insoluble protein polymers. I. Their use for isolation of antigens and antibodies.' J. biol. Chem., 242, 1651. DOONAN, S., DOONAN, H. J., HANFORD, R., VERNON,
C. A., WALKER, J. M., BOSSA, F., BARRA, D., CARLONI, M., FAsELLA, P., RIVA, F. and WALTON,
P. L. (1974). 'The primary structure of aspartate amino transferase from pig heart muscle determined
in part using a protease with specificity for lysine.' FEBS Lett., 38, 229. EDELMAN, G. M., CUNNINGHAM, B. A., GALL, W. E., GOTTLIEB, P. D., RUTISHAUSER, W. and WAXDAL, M. J. (1969). 'The covalent structure of an entire yG molecule.' Proc. nat. Acad. Sci. (Wash.), 63, 78. FRANKLIN, E. C. and PRELLI, F. (1960). 'Structural units of human 7S gamma globulin.'J. clin. Invest., 39, 1933.
Fragmentation of Human IgG GERGELY, J. FUDENBERG, H. H. and VAN LOGHEM, E. (1970). 'The papain sensitivity of IgG myeloma proteins of different heavy chain subclass.' Immunochemistry, 7, 1. GOODMAN, J. W. (196 1). 'Reaction of rheumatoid sera with fragments of papain digested rabbit gamma globulin.' Proc. Soc. exp. Biol. (N.T.), 106, 822. GRAY, W. R. (1972). 'End-group analysis using dansyl chloride.' Methods in Enzymology, volume 25 (ed. by C. H. W. Hirs and S. N. Timasheff), p. 121. Academic Press, London. MCDUFFIE, F. C., OIKAWA, T. and NISHI, I. (1965). 'Reactivity of rheumatoid factor with rabbit yImmunol., 95, 614. globulin.'_3. STANWORTH, D. R. (1960). 'A rapid method for preparing pure serum yG.' Nature (Lond.), 188, 156.
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STELOS, P. (1967). Handbook of Experimental Immunology (ed. by D. M. Weir), p. 4. Blackwell. STEWART, G. A., HUNNEYBALL, I. M. and STANWORTH, D. R. (1975). 'The use of baboon IgG sensitised sheep erythrocytes as an alternative indicator in the study of the interaction of rheumatoid factor with
human IgG.' Immunochemistry, 12, 657. STEWART, G. A., SMITH, A. K. and STANWORTH, D. R. (1973). 'Biological activities associated with the Facb fragment of rabbit IgG.' Immunochemistry, 10, 755. TURNER, M. W. and BENNICH, H. H. (1968). 'Subfragments from the Fc fragment of human yG.' Biochem. J., 107, 171.
Fragmentation of Human IgG by a New Protease Isolated from the Basidiomycete Armillaria mellea I. M. HUNNEYBALL AND D. R. STANWORTH
Department of Experimental Pathology, University of Birmingham
(Received 11 th April 1975; acceptedfor publication 18th April 1975)
Summary. Digestion of human IgG by a new lysine-specific protease, isolated from the basidiomycete Armillaria mellea, produced Fc and Fab fragments similar to those produced by papain digestion of the same molecule. Digestion appeared to be restricted to a single cleavage point within the hinge region of the IgG molecule. Myeloma proteins of IgGl, IgG3 and IgG4 subclasses were found to be digested at an extremely rapid rate whereas IgG2 myeloma proteins appeared to be resistant to digestion by this enzyme. INTRODUCTION Proteolytic degradation of IgG into macromolecular fragments has been widely exploited in studies aimed at defining the location and structural basis of the various biological characteristics demonstrated by antibody molecules. For instance, such an approach has been adopted in the localization of 'auto-antigenic' sites against which are directed the anti-y-globulins found in the sera of patients with rheumatoid arthritis (Goodman, 1961; McDuffie, Oikawa and Nishi, 1965; Stewart, Smith and Stanworth, 1973). Thus, the Fc fragment, which constitutes approximately one third of the IgG molecule, has been shown to possess the binding site(s) for 'general' rheumatoid factors, i.e. those not directed against allotypic determinants (Stewart et al., 1973; Stewart, Hunneyball and Stanworth, 1975). In contrast, both of the fragments produced by plasmin cleavage of IgG (i.e. pFc' and Facb fragments) were found to be unreactive towards these rheumatoid factors. It was of some interest, therefore, to submit IgG to proteolysis by a new lysine-specific protease, recently isolated from the fruiting body of the basidiomycete Armillaria mellea, to ascertain whether it was possible to produce new types of cleavage fragment, particularly originating from the Fc region, which would permit the more precise localization of antigenic sites and other types of effector group. This A. mellea protease has previously been shown to be an endopeptidase which cleaves proteins at peptide bonds N-terminal to lysine residues and, in certain instances, C-terminal to arginine residues (Doonan et al., 1974; Walton, Turner and Broadbent; UK patent number 1263956, 1972).
MATERIALS AND METHODS Materials A. mellea protease was kindly supplied by Dr P. L. Walton (Pharmaceuticals Division, I.C.I. Ltd, Macclesfield). Sheep erythrocytes were obtained from Burroughs Wellcome Correspondence: Dr I. M. Hunneyball, Department of Experimental Pathology, University of Birmingham,
Birmingham BI5 2TJ.
921
I. M. Hunneyball and D. R. Stanworth Ltd, as sheep blood in Alsever's solution. Unless otherwise stated, all general laboratory reagents were obtained as 'Analar' grade from B.D.H. Ltd. Pooled human IgG was isolated from serum by precipitation with 33 per cent saturated ammonium sulphate and then purified by batch ion-exchange chromatography using diethylaminoethyl cellulose (Whatman DE52) (Stelos, 1967; Stanworth, 1960). Human IgGl, IgG2 and IgG3 myeloma proteins were prepared in an identical manner. Human IgG4 myeloma protein was isolated by ion-exchange chromatography on a column (90 x 2-2 cm) of DEAE-cellulose (Whatman DE52) equilibrated with 0-01 M phosphate buffer, pH 6-3, by elution with a sodium chloride gradient (0.001-0-05 M), IgG4 eluting at a position corresponding to 0-02 M sodium chloride. Purity of the IgG preparations was assessed by immunoelectrophoresis and analytical ultracentrifugation at a concentration of 10 mg/ml. In addition, the myeloma proteins were found to be pure by both immunodiffusion using subclass specific antisera and Gm typing. The Fc and Fab fragments of human IgG were prepared by digestion of IgG with papain (Sigma Chemical Company, Ltd) in the presence of cysteine and purified by gel-filtration on Sephadex G-150 (Pharmacia Ltd), followed by ion-exchange chromatography on carboxymethyl Sephadex (Sephadex C50, Pharmacia Ltd) and diethylaminoethyl cellulose (Whatman DE52) according to Franklin and Prelli (1960).
922
Antisera Sheep antisera to human IgG, human Faby fragment, and the y chain were kindly provided by Dr D. Catty (Department of Experimental Pathology, University of Birmingham). Antiserum to the Fc fragment was prepared by absorption of the sheep antihuman y chain antiserum with pure Fab fragment. Specific anti-Cy2 domain antiserum was obtained by absorbing sheep anti-Fc antiserum with pFc' fragment (Cy3) domain, prepared according to Turner and Bennich (1968), which had been insolubilized using ethyl chloroformate according to Avrameas and Ternynck (1967). Antiserum to the Cy3 domain was prepared by injecting guinea-pigs with pFc' fragment in Freund's complete adjuvant. Baboon antiserum to sheep erythrocytes was kindly supplied by Mr G. A. Stewart (Department of Experimental Pathology, University of Birmingham). Rheumatoid arthritis sera were obtained from the Queen Elizabeth Hospital (Birmingham) by courtesy of Dr C. F. Hawkins.
Immunoelectrophoresis and immunodifusion Immunoelectrophoresis was performed in a Shandon electrophoresis apparatus using 1 per cent Oxoid Agar number 3 in barbitone buffer, pH 8-6 (I = 0.05). Electrophoresis was carried out at a constant current of 18 mA per plate for 1-5 hours. Immunodiffusion was allowed to proceed for 24 hours at 4V.
N-terminal analysis N-terminal analysis was performed according to the method of Gray (1972) employing dansyl chloride. Reactivity with 'general' rheumatoidfactors The reactivity of human IgG subfragments with 'general' rheumatoid factors was measured by a haemagglutination-inhibition technique employing diluted rheumatoid
923 Fragmentation of Human IgG92 serum and sheep erythrocytes sensitized with a subagglutinating dose of baboon anti-sheep erythrocyte antiserum according to Stewart et al. (1975). Prior to testing, the rheumatoid sera were clarified by centrifugation, decomplemented by incubation at 560 for 20 minutes and absorbed with an equal volume of packed sheep erythrocytes. The rheumatoid serum was diluted with 0*0l m phosphate-buffered saline (0.15 M), pH 7-2, to give an agglutination titre of 1 in 4 with sensitized erythrocytes. To serial doubling dilutions (0-025 ml) of inhibitor (at an initial concentration of 10 mg/ml) in the wells of a microtitre tray was added an equal volume of the diluted rheumatoid serum. After incubation for 1 hour at 370, an aliquot (0-025 ml) of a 1 per cent suspension of sensitized cells was added to each well. The inhibition titres were read after incubation for a further hour.
Gm typing The expression of allotypic markers on the subfragments was determined by a haemagglutination-inhibition technique employing human 0 Rh'+ erythrocytes sensitized with incomplete anti-D antibodies of known Gm type and sera from normal transfused blood donors. RESULTS DIGESTION CONDITIONS
Pooled normal human JgG (20 mg/ml) in 0-0l m Tris-HC1 buffer, pH 7-0, containing 0 -l15 m sodium chloride and 0*002 m calcium acetate, was digested with A. mellea protease at an enzyme: substrate ratio of 1 :1I00 (wt/wt) at 3 7'. Serial samples were removed at intervals between 10 seconds and 24 hours and the enzyme inactivated by addition of an equal volume of 0-0l m disodium. EDTA in 0-01 m phosphate-buffered saline (0-l5 m), pH 7-2. Immunoelectrophoresis, using sheep anti-IgG antiserum, indicated that digestion had occurred almost instantaneously and that no further increase in digestion was evident even after 24 hours. This finding was substantiated by gel filtration analysis on a column (90 x 2-2 cm) of Sephadex G-150 equilibrated with 0'05 m ammonium carbonate buffer, pH 8 -6. The optimum digestion time was found to be 3 hours using an enzyme : substrate ratio of 1:1I000 (wt/wt). ISOLATION OF FRAGMENTS
The digestion products were separated on a column (90 x 3 -2 cm) of Sephadex G- 150 equilibrated with 0-05 m ammonium carbonate buffer, pH 8-6. Three major protein fractions could be distinguished from the elution profile (Fig. 1): fraction 1 eluted in a position corresponding to undigested IgG; fraction 2 eluted in a position similar to papain Fc and Fab fragments; fraction 3 contained dialysable material. Fractions 1 and 2 were purified by rechromatography on a column (90 x 2 -2 cm) of Sephadex G- 150 equilibrated with 0-05 m ammonium carbonate buffer, pH 8-6, and characterized by immunoelectrophoresis against a variety of antisera (Fig. 2a). Fraction 1 consisted of a single component, precipitating with both anti-Fc and anti-Fab antisera, with an electrophoretic mobility identical to that of native human IgG. Fraction 2 contained two components, exhibiting non-identity against sheep anti-JgG antiserum, with electrophoretic mobilities similar to those of the papain Fc and Fab fragments. The Fc-like fragment precipitated with the I
924
L M. Hunneyball and D. R. Stanworth Or-
/I' I)
E
\
I
0
0-5 0
0
X,1 I \ L
o00
L
200 300 Elution volume (ml)
\
3
400
FIG. 1. Gel filtration of a 3-hour A. mellea protease digest of pooled human IgG on a column (90 x 3-2 cm) of Sephadex G-150 equilibrated with 0 05 ammonium carbonate buffer, pH 8-6.
FIG. 2 (a) Immunoelectrophoresis of Sephadex G-150 fractions 1 and 2 of a 3-hour A. mellea protease digest of human IgG, using sheep anti-IgG (A), sheep anti-Fc (B), and sheep anti-Fab (C) antiserum. Native IgG (N) was included as a control. (b) Immunoelectrophoresis of: (1) papain Fc fragment; (2) A. mellea protease fraction 2(2); (3) A. mellea protease fraction 2(1); (4) papain Fab fragment (using sheep anti-IgG antiserum). (c) Gel diffusion analysis of: (1) papain Fc fragment; (2) A. mellea protease fraction 2(2); (3) A. mellea protease fraction 2(1); (4) papain Fab fragment (using sheep anti-IgG antiserum (A). Well (5) contained buffer only.
anti-Fc antiserum but not the anti-Fab antiserum; whereas the Fab-like fragment precipitated with the anti-Fab antiserum but not the anti-Fc antiserum (Fig. 2a.) Human IgG1 myeloma proteins were found to behave in an identical manner to pooled human IgG on treatment with A. mellea protease. However, unless otherwise stated, normal pooled human IgG was used. Due to the similarity between the fraction 2 components and papain Fc and Fab fragments, separation of the fraction 2 components was achieved by the procedure previously adopted for the separation of the papain fragments, i.e. ion-exchange chromatography on carboxymethyl Sephadex and diethylaminoethyl cellulose by the method of Franklin and Prelli (1960) as outlined in Fig. 3. Fraction 2 was dialysed exhaustively
925 Fragmentation of Human IgG against 0*01 M phosphate buffer, pH 7-6, and applied to a column (60 x 2-2 cm) of CM Sephadex (C50, Pharmacia Ltd) equilibrated with the same buffer. As will be seen from Fig. 4a, two fractions were resolved by stepwise elution with 0-01 M phosphate buffer, pH 7-6 (fraction 2A) and 0 3 M phosphate buffer, pH 7-6 (fraction 2B). Fraction 2A was concentrated, dialysed against 0-01 M phosphate buffer, pH 8-0, and applied to a column (60 x 2-2 cm) of DEAE-cellulose (Whatman DE-52) equilibrated with the same buffer. As will be seen from Fig. 4b, two fractions were resolved by stepwise elution with 0.01 M phosphate buffer, pH 8-0 (fraction 2A(1)) and 0-3 M phosphate buffer, pH 8-0 (fraction 2A(2)). Fraction 2B was purified in a identical manner giving rise to two fractions: 2B(1) and 2B(2) as shown in Fig. 4c. Fractions 2A(1) and 2B(1) were pooled and designated 2(1). Similarly, fractions 2A(2) and 2B(2) were pooled and designated 2(2). Human IgG Am protease, pH 7 0
(1:1000enzyme: substrate) 3 hours at 370C Sephadex G-150 3
2
(Peptides)
(IgG) CM Sephadex pH 76
03 M
|O.I M
2B DEAE-cellulose pH 8-0
2A DEAE-cellulose pH 8-0
00I M
01 3M
2A(1)
2A(2)
2(1)
OCOI M 2B(l)
0 3M
2B(2)
2(2)
FIG. 3. Outline of purification scheme for A. mellea protease subfragments of pooled human IgG.
Both fractions appeared pure by immunoelectrophoresis at a concentration of 5 mg/ml using sheep anti-IgG antiserum (Fig. 2b). However, slight contamination of fraction 2(1) with fraction 2 (2), and vice versa, was evident on gel diffusion analysis of the two fractions at 5 mg/ml against the same antiserum (Fig. 2c). CHARACTERIZATION OF THE FRAGMENTS
On immunoelectrophoresis (Fig. 2b) fraction 2(1) demonstrated similar mobility to the papain Fab fragment. In contrast, fraction 2(2) was found to be slower electrophoretically than the papain Fc fragment. The results of gel diffusion analyses of the purified fractions are summarized in Table 1. Fraction 2 (1) gave a reaction of complete identity with a preparation of the papain Fab fragment on gel diffusion against anti-IgG (Fig. 2c) and anti-Fab antisera. Fraction 2 (2) gave a reaction of complete identity with a preparation of the papain Fc fragment on gel diffusion against anti-IgG, anti-Fc, anti-Cy2 domain and
I. M. Hunneyball and D. R. Stanworth
926
(a) I'
1I0
0-5 -
0
1-0
0.3 M
\
001 M /
/,L_
100 b)
200
-1
-
s400
2B
!_ 500
I'\F"
I\
E
a05
0 0-5
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-
(f2AI>-| t-.122,-
0
co CIQ~~~~~~C 100
200 300
40
50
400
500
I' I 0.01 M 100
c
200
0.3M/ 300
Elution volume (ml)
FIG. 4. (a) Purification of Sephadex G150 fraction 2 on a column (60x2 2 cm) of carboxymethyl Sephadex (C50) equilibrated with 0-01 M phosphate buffer, pH 7-6. Fraction 2A was eluted with 0-01 M phosphate buffer, pH 7-6. Fraction 2B was eluted with 0-3 M phosphate buffer, pH 7-6. (b) Purification of CM Sephadex fraction 2A on a column (60 x 2-2 cm) of DEAE-cellulose (Whatman DE52) equilibrated with 0-01 M phosphate buffer, pH 8-0. Fraction 2A(1) was eluted with 0-01 M phosphate buffer, pH 8f0. Fraction 2A(2) was eluted with 0 3 M phosphate buffer, pH 8-0. (c) Purification of CM Sephadex fraction 2B on a column (60 x 2-2 cm) of DEAE-cellulose (Whatman DE52) equilibrated with with 0-01 M phosphate buffer, pH 8-0. Fraction 2B(l) was eluted with 0-01 M phosphate buffer, pH 8-0. Fraction 2B(2) was eluted with 0 3 M phosphate buffer, pH 8-0. TABLE 1 GEL-DIFFUSION PRECIPITIN ANALYSIS OF PURIFIED PREPARATIONS OF A. mellea PROTEASE DIGESTION FRAGMENTS OF POOLED HUMAN IgG (FRACTIONS
2(1)
AND
2(2)) USING VARIOUS SPECIFIC ANTISERA
Antiserum
Fraction 2 (1)
Fraction 2 (2)
Sheep anti-IgG Sheep anti-Fab Sheep anti-Fc Sheep anti-Cy2 Guinea-pig anti-Cy3
+ + -
+ + + +
927 Fragmentation of Human IgG anti-Cy3 domain antisera. Analytical ultracentrifugation indicated that both fractions sedimented at a similar rate to the papain Fc fragment (Table 2). N-terminal analysis of fraction 2(2) which had been prepared from an IgG1 myeloma protein, indicated the presence of an N-terminal lysine residue. This contrasted with the presence of a threonine residue at the N-terminus of the papain Fc fragment of the same myeloma protein. TABLE 2 SEDIMENTATION COEFFICIENTS OF PURIFIED A. mellea PROTEASE DIGESTION FRAGMENTS OF POOLED HUMAN IgG (FRACTIONS 2(1) AND 2(2)) AT A CONCENTRATION OF 4 mg/ml IN 0-01 M PHOSPHATE-BUFFERED SALINE (0.15 M), pH 7-2
Sample
S20,w
Pooled human IgG Papain Fc fragment Fraction 2(1) Fraction 2 (2)
6-82 + 0 05 3-92 + 0)04 3-74+0 06 3 85+ 0 06
SUBFRAGMENTATION OF THE Fc FRAGMENT
Subfragmentation of papain Fc fragment by A. mellea protease was attempted. Pooled human IgG Fc fragment (20 mg/ml) in 0-01 M Tris-HCl buffer, pH 7 0, containing 0-15 M sodium chloride and 0-002 M calcium acetate, was digested with A. mellea protease at an enzyme:substrate ratio of 1:100 (wt/wt) at 370. Serial samples were removed at intervals between 10 seconds and 48 hours and the enzyme inactivated by addition of an equal volume of 0-01 M disodium EDTA in 0 01 M phosphate-buffered saline (0.05 M), pH 7-2. The samples were analysed by immunoelectrophoresis, using sheep anti-Fc antiserum. Evidence of some proteolysis was apparent after 6 hours, but even after 24 and 48 hours digestion the extent of cleavage indicated by immunoelectrophoretic analysis was very limited. This conclusion was confirmed by analytical gel filtration of the 24- and 48-hour digests on a column (90 x 2-2 cm) of Sephadex G-100 equilibrated with 0 05 M ammonium carbonate buffer, pH 8-6 (Fig. 5). Thus it would appear that under the conditions em0*75
E
-
0 50 -l
i
co'1
0
O 025
II
0
100
I\
. ' 200
300
400
Elution volume (ml)
FIG. 5. Gel filtration of a 48-hour A. mellea protease digest of human IgG Fc fragment on a column (90 x 2-2 cm) of Sephadex G-100 equilibrated with 0 05 M ammonium carbonate buffer, pH 8-6.
I. M. Hunneyball and D. R. Stanworth 928 ployed for fragmentation of IgG, the papain Fc fragment is essentially resistant to digestion by A. mellea protease.
DIGESTION OF IgG SUBCLASSES
Preliminary investigations into the susceptibility of the four subclasses of human IgG by A. mellea protease were performed. IgG myeloma proteins (10 mg/ml) in 0 01 M phosphate buffer, pH 7-0, containing 0*15 M sodium chloride and 0-002 M calcium acetate were digested at an enzyme:substrate ratio of 1:100 (wt/wt) at 370 for 30 minutes. Excess solid disodium EDTA was added to terminate digestion and the digests analysed by immunoelectrophoresis (Fig. 6). Control myeloma proteins were treated in an identical manner but in the absence of enzyme. IgG1 and IgG3 myeloma proteins seemed to be completely digested by the A. mellea protease, whereas IgG4 appeared to be only partially digested. In contrast, IgG2 myeloma protein seemed to be resistant to digestion.
FIG. 6. Immunoelectrophoresis of A. mellea protease digests of myeloma proteins of the four subclasses of human IgG, using sheep anti-IgG antiserum. Number denotes subclass of the myeloma protein. AUTO-ANTIGENICITY OF THE FRAGMENTS
The capacity of the purified subfragments of pooled IgG to react with 'general' rheumatoid factors (i.e. those not directed against allotypic determinants) was investigated, the results of which are summarized in Table 3. Fraction 2 (1) showed no reactivity towards TABLE 3 CAPACITY OF A. mellea PROTEASE DIGESTION FRAGMENTS OF POOLED HUMAN IgG TO INHIBIT THE AGGLUTINATION, BY RHEUMATOID ARTHRITIS SERUM, OF SHEEP ERYTHROCYTES SENSITIZED WITH BABOON IgG. (THE RHEUMATOID ARTHRITIS SERUM WAS DILUTED TO GIVE A TITRE OF 1 IN 4 WITH SHEEP ERYTHROCYTES SENSITIZED WITH BABOON IgG)
Sample IgG Fraction 2 (1) Fraction 2 (2) Papain Fc Papain Fab
Lowest concentration (mg/ml) of protein or fragment giving inhibition
0-6 >5 0
03 03 >5 0
929 Fragmentation of Human IgG 'general' rheumatoid factors whereas fraction 2(2) reacted to the same extent as the papain Fc fragment. The localization of Gm and InV allotypic markers on the purified subfragments of pooled IgG was also investigated; fraction 2(1) expressed the Gm (f) and InV (1) markers, whereas fraction 2(2) expressed Gm (a), (x), (b1) and (b4) markers. DISCUSSION ofhuman mellea Cleavage IgG byA. protease was found to proceed at a muchfaster rate than that by other common proteolytic enzymes such as papain and pepsin. Similar kinetics have been demonstrated for the action of this enzyme on casein and fibrinogen (Walton, personal communication); moreover, the action of A. mellae protease on these proteins produced large fragments relatively resistant to further digestion by the enzyme, a feature exhibited by the enzyme in its action on native IgG and substantiated by the resistance of the papain Fc fragment to digestion by the enzyme. The products ofA. mellea protease digestion of human IgG appear to be similar immunochemically to the Fc and Fab fragments produced by papain digestion of the same molecule. The Fab-like fragment produced by A. mellea protease digestion (fraction 2(1)) proved to be identical to the papain Fab fragment by all criteria applied. The Fc-like fragment produced by A. mellea protease digestion (fraction 2(2)) was identical to the papain Fc fragment by gel diffusion analysis with specific heteroantisera, analytical'ultracentrifugation, and its capacity to react with 'general' rheumatoid factors; but it was electrophoretically slower than the papain Fc fragment and contained lysine at the N-terminus in contrast to the N-terminal threonine of the papain Fc fragment. The slower electrophoretic mobility of fraction 2(2) indicates a higher content of basic residues which would be consistent with cleavage of IgGl by A. mellea protease at the Asp-Lys bond at position 221-222 within the hinge region of the molecule (Fig. 7) (assuming the sequence of the IgGi myeloma protein Eu described by Edelman, Cunningham, Gall, Gottlieb, Rutishauser and Waxdal, 1969). Thus fraction 2(2) would differ from the papain Fc fragment by only three amino acid residues at the N-terminus of the fragment. This proposed similarity between fraction 2(2) and the papain Fc fragment is supported by the resistance of both fragments to subsequent digestion by A. mellea protease. Am protease
N
L
)
Papain
II
S N
S
N
sA
by
S~~ ~
SI
Y
C
~~~ I r
I~~~~
s
C
N
FIG. 7. Probable location of the point of cleavage of IgG by A. mellea protease. Residue numbering is based on the sequence of the IgGl myeloma protein Eu (Edelman et al., 1969).
930 I. M. Hunneyball and D. R. Stanworth Cleavage by A. mellea protease at the hinge region of IgG, is consistent with the observed expression of allotypic markers Gm (a), (x), (b') and (b4) on the Fc-like fragment and of Gm (f) and InV(l) on the Fab-like fragment. Cleavage of peptide bonds N-terminal to lysine residues is in accord with the reported primary specificity of A. mellea protease for peptide bonds within other proteins, such as casein, fibrinogen and aspartate aminotransferase (Doonan et al., 1974; Walton, personal communication). Therefore, the cleavage of IgG by A. mellea protease appears to be similar in all respects to the reported mode of action of the enzyme on other protein molecules. The limited specificity of A. mellea protease with regard to cleavage of human IgG appears to parallel the restricted specificity of plasmin for the hinge region of the molecule; the difference in specificity between the two enzymes being reflected by the position of the bond cleaved relative to the lysine residue, i.e. plasmin cleaves peptide bonds C-terminal to lysine, whereas A. mellea protease cleaves peptide bonds N-terminal to lysine residues. A. mellea protease appeared to cleave IgG3 and IgG4 in a similar fashion to IgGl, producing two subfragments. The susceptibility of the different subclasses to cleavage by the enzyme was found to be similar to the reported susceptibility of the proteins to digestion by papain (Gergely, Fudenberg and Van Loghem, 1970). In contrast to papain, however, the difference in susceptibility to digestion by A. mellea protease between the subclasses was found to be more definitive, i.e. IgGl and IgG3 appeared to be digested completely to Fc and Fab fragments, whereas no trace of digestion of IgG2 was evident under the same conditions. This finding indicates the potency of this enzyme for subclass determination of myeloma proteins. A. mellea protease appears to be an enzyme of great potential for producing large subfragments from protein molecules for studies of the submolecular localization of various biologically active sites. With regard to cleavage of human IgG, this protease can rapidly produce Fc and Fab fragments in high yield, due to the resistance of these subfragments to further degradation by the enzyme. The increased size of the A. mellea protease Fc fragment in comparison with that produced by papain cleavage may be of considerable use in the localization of hinge region-located effector sites. However, from this study, it was not possible to gain any further information with regard to the localization of the 'general' rheumatoid factor-reactive determinants within the Fc fragment. ACKNOWLEDGMENTS The Gm typing of the myeloma proteins and fragments was kindly performed by Miss D. F. Barr (Regional Blood Transfusion Service). Generous financial support from the Medical Research Council is gratefully acknowledged. REFERENCES
AVRAMEAS, S. and TERNYNCK, T. (1967). 'Biologically active water-insoluble protein polymers. I. Their use for isolation of antigens and antibodies.' J. biol. Chem., 242, 1651. DOONAN, S., DOONAN, H. J., HANFORD, R., VERNON,
C. A., WALKER, J. M., BOSSA, F., BARRA, D., CARLONI, M., FAsELLA, P., RIVA, F. and WALTON,
P. L. (1974). 'The primary structure of aspartate amino transferase from pig heart muscle determined
in part using a protease with specificity for lysine.' FEBS Lett., 38, 229. EDELMAN, G. M., CUNNINGHAM, B. A., GALL, W. E., GOTTLIEB, P. D., RUTISHAUSER, W. and WAXDAL, M. J. (1969). 'The covalent structure of an entire yG molecule.' Proc. nat. Acad. Sci. (Wash.), 63, 78. FRANKLIN, E. C. and PRELLI, F. (1960). 'Structural units of human 7S gamma globulin.'J. clin. Invest., 39, 1933.
Fragmentation of Human IgG GERGELY, J. FUDENBERG, H. H. and VAN LOGHEM, E. (1970). 'The papain sensitivity of IgG myeloma proteins of different heavy chain subclass.' Immunochemistry, 7, 1. GOODMAN, J. W. (196 1). 'Reaction of rheumatoid sera with fragments of papain digested rabbit gamma globulin.' Proc. Soc. exp. Biol. (N.T.), 106, 822. GRAY, W. R. (1972). 'End-group analysis using dansyl chloride.' Methods in Enzymology, volume 25 (ed. by C. H. W. Hirs and S. N. Timasheff), p. 121. Academic Press, London. MCDUFFIE, F. C., OIKAWA, T. and NISHI, I. (1965). 'Reactivity of rheumatoid factor with rabbit yImmunol., 95, 614. globulin.'_3. STANWORTH, D. R. (1960). 'A rapid method for preparing pure serum yG.' Nature (Lond.), 188, 156.
931
STELOS, P. (1967). Handbook of Experimental Immunology (ed. by D. M. Weir), p. 4. Blackwell. STEWART, G. A., HUNNEYBALL, I. M. and STANWORTH, D. R. (1975). 'The use of baboon IgG sensitised sheep erythrocytes as an alternative indicator in the study of the interaction of rheumatoid factor with
human IgG.' Immunochemistry, 12, 657. STEWART, G. A., SMITH, A. K. and STANWORTH, D. R. (1973). 'Biological activities associated with the Facb fragment of rabbit IgG.' Immunochemistry, 10, 755. TURNER, M. W. and BENNICH, H. H. (1968). 'Subfragments from the Fc fragment of human yG.' Biochem. J., 107, 171.
