Location of immunoglobulin cytophilic activity

Eur. J. Immunol. 1976.6: 101-107 M.D. Alexander, R.G.Q. Leslie and S. Cohen Department of Chemical Pathology, Guy’s Hospital Medical School, London

101

Cytophilic activity of enzymatically derived fragments of guinea pig lgG2 The hydrolysis products from short term exposure of guinea pig IgG2 t o papain and pepsin have been characterized. Papain hydrolysis liberates 4 types of Fc fragment, only one of which retains both an interchain disulfide bond and an intact c H 2 domain, cFc, mol. wt. 56 000. The other three fragments noncovalently linked F c (nFc) (mol. wt. 56 000), incomplete Fc (iFc) (mol. wt. 3 9 000) and Fc’(23 000) represent further degradation products of covalently linked complete F c (cFc). The cytophilic activities of these fragments as well as F(ab‘), and pFc’ from pepsin hydrolysis, were studied t o determine the domain(s) responsible for binding to homologous peritoneal macrophages. Only the native immunoglobulin and the intact cFc manifested cytophilic activity; in particular pepsin-derived pFc’ and Fc‘ were inactive. Following mild reduction and alkylation, performed t o affect only the interchain disulfide bonds, the cytophilic activity of cFc was markedly reduced. The low cytophilic activity in the pFc’ fragment suggests that the C H 2 domains play a major part in binding to the macrophage Fc receptor through a site(s) stabilized by the interchain disulfide bonds.

1. Introduction

The domain hypothesis proposed by Edelman [ 11 and its confirmation through x-ray crystallographic and sequence studies [2, 31 have provided a theoretical and practical framework t o investigate immunoglobulin (Ig) structure and function. Edelman [ 1 ] proposed that each domain would mediate at least one Ig function. The variable domains of both heavy and light chains contain the hypervariable sections of sequence which participate directly in t h e interaction with antigen [4]. Other activities are mediated by the constant domain of the heavy chains CH 1 CL, CH 2 and CH 3. Reid [ 51 and Sandberg et al., [6] showed that F(ab’):! fragments of rabbit and guinea pig IgG respectively, retain the ability to activate the alternate complement pathway. It is, however, not clear whether the hinge region or the CH 1 CL domain is involved in this effector function. A fragment from the CH 2 domain of mouse IgG2, characterized by Kehoe e t al. [7, 81 retains low complement binding activity. This involvement of the c H 2 domain of human IgGl and rabbit IgG in the classical complement pathway has been confirmed by Ellerson et al. [9] and Colomb and Porter [ 101. The importance of the C H domain ~ in the interaction of IgG with F c receptors during antibody-dependent cytotoxicity was demonstrated by Wislq4ff et al. [ 1 11 using human lgG3

[I 12531

Correspondence: Michael David Alexander, Department of Chemical Pathology, Guy’s Hospital Medical School, London SE1 9RT, England Abbreviations: cFc: Covalently linked complete Fc nFc: Noncovalently linked Fc iFc: Noncovalently linked incomplete Fc Fch: Fragment from human IgC3 comprising complete Fc plus the hinge region pFc’: Pepsin-derived fragment comprising two noncovalently linked, intact C H domains ~ Fc’: Papain-derived fragment comprising two noncovalently linked peptides with heterogeneous carboxy terminal residues from the C H domains ~ PCA: Passive cutaneous anaphylaxis BGG: Bovine IgG FCA: Freund’s complete adjuvant PBS: Phosphate buffered saline DTT: Dithiothreitol FTH: Phenylthiohydantoin TCM 199: Tissue culture medium 199

F c and Fch fragments. The Fch fragment was more active than the native Fc and both fragments were inactivated by mild reduction and alkylation. Early studies o n the biological activity of pFc‘ and Fc’ fragments of rabbit and human IgG indicated that the c H 3 domain was not involved in passive cutaneous anaphylaxis (PCA) and complement fixing activities [ 1 2- 141. Recently, however, Minta and Painter [ 151 have obtained indirect evidence for the involvement of the c H 3 domain in PCA reactions. The same domain has also been shown t o mediate macrophage cytophilic activity of human IgG [ 16, 171. The products of papain and pepsin hydrolysis of guinea pig IgG2 [ 181 have been further characterized in the present study. By using these products in conjunction with a direct binding assay for macrophage cytophilic antibody [ 191 we have attempted t o locate the domain of guinea pig IgG2 responsible for mediating this effector function. I n order t o obtain sufficient quantities of IgG2, the sera from immunized guinea pigs was used as the source. Rigorous precautions were taken t o exclude immune complexes and aggregates before measuring the cytophilic activity [ 201 and preparing enzymatically derived fragments.

2. Materials and methods 2.1. Animals and immunization

Hartley strain guinea pigs (300-700 g) were used. Guinea pig anti-bovine IgG (BGG) antisera were raised as described by Leslie and Cohen [ 201. Briefly, animals were injected o n days 1 and 8 with BGG (0.5 mg) in Freund’s complete adjuvant (FCA) (0.5 ml) administered intramuscularly and subcutaneously, and boosted o n day 2 1 by intraperitoneal injection of alumina adsorbed antigen. The animals were bled by cardiac puncture o n days 28 and 35 and subsequently at 6 week intervals, 7 days after intraperitoneal rechallenge. Antisera to guinea pig IgC,, Faby, and Fcy2 were raised in rabbits, employing the same immunization protocol. All were stored at -30 ‘C.

102

M.D. Alexander, R.G.Q. Leslie and S. Cohen

2.2. Preparation of IgGz

Eur. J. Immunol. 1976.6: 101-107 2.6. Physical and chemical characterization of Fc fragments

Molecular weights of the F c fragments from papain and peptic IgG, was isolated from pooled, euglobulin free, antisera by digestion were determined by gel filtration of the totally resalt precipitation and anion exchange chromatography as deduced and alkylated polypeptide chains through Sephadex scribed previously [ 19, 221. The IgG2 was passed through upG-200 in 5 M guanidinium chloride [ 181. Dextran blue, ward flow Sephadex G-200 columns in 0.05 M Tris HC1,O. 1 M NaCl, pH 8.2; t h e 7 S pool was recovered and stored at - 3 0 "C. y2-chains, L-chains, lysozyme and tyrosine were employed as standard markers. Amino acid analyses were performed Radio-iodination of IgG2 was performed as described [ 191. o n a Locarte analyzer as described previously [22]. Carboxy terminal analysis was carried out using hydrazinolysis and was performed by the method of Bradbury [24] and Fraenkel 2.3. Preparation of papain and pepsin hydrolysis products of Conrat and Tsung [25] and is described fully in Leslie et al. [ 181. IgGz Amino acid sequence determination was performed on samples Peptic and papain digestion of IgG2 has been described in an which had been totally reduced with a twofold molar excess earlier paper [ 181. F(ab'), and pFc' from peptic digestion were of DTT in 1 M Tris HCl, pH 8.2, in 7 M guanidinium chloride separated by gel filtration o n Sephadex G-200. The Fab and F c under N2 for 1 h at 37 'C, alkylated with [3H]iodoacetic acid products from papain digestion were isolated by anion exchange (2.4 M excess) at room temperature for 1 h, and desalted in chromatography o n DEAE-cellulose (Whatman DE32) with 10 5% acetic acid. Sequencing of the first 6 residues of each 50 mM Tris HC1, pH 8.2, and a linear salt concentration grafragment was kindly performed by Dr. M.D. Waterfield using dient (0-0.4 M NaCl) using an 11 3 0 Ultrograd (LKB Producter, a Beckman 890C sequencer and an 0.1 M Quadrol program Stockholm) gradient mixer. High resolution of the F c peptides [ 261. Identification of S-carboxymethyl cysteine was accomwas achieved by arresting the gradient during peak elution plished by scintillation counting of a 4 % aliquot of each step A:&, > 0.2) using a Uvicord 111 flow-through recorder t o on Nuclear Chicago Mark I liquid scintillation counter. Quantitative identification of each step was performed by HI hydromonitor the elution profile and control the gradient mixer. lysis of the phenylthiohydantoin (PTH) derivatives [ 271 and Under these conditions, F a b elutes predominantly with the pregradient buffer, while the Fc peptides elute in the 0.05amino acid analysis of the hydrolyzates. Recoveries were nor0.25 M NaCl range. For some experiments a complete F c malized t o an internal PTH norleucine standard ( 2 5 nmoles) pool was recovered by immunoadsorption of the whole paintroduced after cyclization of the original cleavage products. pain digest with Sepharose-bound rabbit anti-guinea pig Faby (see below). 2.7. Macrophage-binding assay

2.4. Immunoadsorption of isolated digest fragments

Solid phase immunoadsorbents were prepared by coupling the IgG fraction from rabbit antisera specific for either guinea pig Fcy, or F a b t o Sepharose 4B (Pharmacia, Uppsala, Sweden). Rabbit IgG was prepared by precipitation of the serum with ammonium sulfate at 50 % saturation and ion exchange chromatography through DEAE-cellulose (Whatman DE22) equilibrated with 0.021 M disodium phosphate, 0.005 M p c ~ tassium dihydrogen phosphate, pH 7.5. The rabbit IgG was covalently coupled t o Sepharose 4B using cyanogen bromideactivated Sepharose in the following proportions: 50 mg CNBr: 1 ml Sepharose 4B: 1-2 mg IgG at pH 6.5 [23]. F(ab'), and Fab fragments were freed of Fc contamination by passage through columns containing IgG antibody specific for guinea pig Fey, Fc preparations were similarly purified by elution through a column containing IgG antibody t o Fabyz. Immunoadsorption was carried out a t 4 'C in PBS, pH 6.9.

2.5. Reduction and alkylation of F c fragments Reduction was carried out in 0.5 M Tris HC1, pH 8.5, with dithiothreitol (DTT) at between 2 and 100 mM for 45 min at 22 'C. Alkylation was achieved by adding a 1 0 % molar excess of iodoacetamide at 4 'C for 30-40 min. Excess iodoacetamide was removed by extensive dialysis against PBS, pH 7.4. Total reduction and alkylation of F c fragments has been described previously [ 181.

All protein samples were subjected t o gel filtration o n G-200 upward flow columns in 50 mM Tris HCl, pH 8.2, 100 mM NaCI, and ultracentrifugation at 400 000 x g for 90 min to remove aggregated material above 20 S value. The assay employed was essentially that described by Leslie and Cohen [ 191. In each experiment, series of tubes containing 0.5 ml of 7 S1251-labeled IgG, ( 2 pg), 0.5 ml of IgG2 of Ig fragment (1 5-200 pg) and 0.1 ml of peritoneal exudate cells ( - 3 x lo6 macrophages) were incubated with shaking at room temperature for YO min. The cells were washed twice with cold TCM199 (1 ml), the bound label in each tube was counted and was expressed as a decimal fraction of the total '251-labeled IgGz in the incubation mixture. To facilitate comparison of the inhibitory activities of different fragments, determined in separate experiments, IgG2 was included as an internal control in each experiment and the data were normalized as follows. The inhibitory data for IgG2 in each assay were employed t o construct a Scatchard plot [ 28, 191 and the limiting y-value (maximum fraction bound) was determined. This value usually approximated t o the observed fractional uptake of '251-labeled IgG2 in the absence of inhibitor. The '251-labeled IgG2 binding in the presence of either inhibitor IgG2 or fragment was then expressed as a percentage of the limiting y-value and a second set of Scatchard-type plots was derived by plotting (% of maximum binding) versus (5% of maximum binding x molar concentration of inhibitor IgG, or fragment). The inhibition slopes for each fragment could then be expressed as a percentage of the inhibition slope for IgGz to give a relative binding activity. Replicate assays were performed o n most fragments and the mean slopes S.D. have been determined. Standard deviation and linear regression analyses were performed using a Monroe 1930 calculator.

*

Location of immunoglobulin cytophilic acitvity

Eur. J. Immunol. 1976.6: 101-107

103

3. Results

3.1.2. The fragments from papain hydrolysis

3.1. Separation and characterization of IgGz hydrolysis fragments

IgG2 was digested with papain and the products separated by anion exchange chromatography. The pre-gradient and gradient peaks were pooled as shown in Fig. 1. Each pool was freed of 3 S material by passage through Sephadex G-1 00 in PBS and the Fc fragments in the gradient pools were purified by immunoadsorption with Sepharose-bound rabbit anti-Faby2. Three major species of F c fragment were distinguishable: a) intact Fc, (Pools G1 and G2) possessing all the class-specific determinants, with an approximate molecular weight of 5 x 104, and y2 electrophoretic mobility; b) a smaller, more acidic Fc fragment (pool G3, mol. wt. 3.5 x 1 04), which was antigenically indistinguishable from (a) and, finally, c) an Fc’ fragment (pool G4, mol. wt. 2.1 x 104) with 0 mobility, lacking many of the class-specific determinants, and displaying antigenic identity with pFc’ (see Table 2). Passage of intact Fcy2 (G1 + 2 pool) through Sephadex G-100 in 20 % acetic acid revealed the presence of both covalently linked F c dimer peptides (cFc), and monomeric peptides (nFc). Under similar conditions, the smaller F c fragment (iFc, G 3 pool) dissociated into roughly equimolar amounts of F c monomer and Fc’, indicating that, in the native state, the fragment is a mixed molecule containing both these components. Fc’ (G4 pool) elutes as a single symmetrical peak under these conditions (Table 2).

>

3.1.1. The fragments from peptic hydro!ysis

(Fab’), and pFc‘ fragments were prepared by peptic digestion and gel filtration, followed by immunoadsorption with Sepharose-bound rabbit anti-Fc and anti-Fab antibodies respectively. Previous characterization [ 181 has shown (Fab’), t o be a 5 S dimeric fragment, indistinguishable antigenically from Fab and totally lacking Fc determinants. The pFc’ fragment lacks some of the class-specific (Fc) determinants of IgG2 but possesses the same C-terminal glycine residue as the intact 72-chain [ 181. The molecular weight of monomeric pFc’ was estimated as 13 000 f 400 by gel filtration of the totally reduced and alkylated fragment in 5 M guanidinium chloride [ 181. Under normal conditions pFc’ behaves as a dimer (mol. wt. 26 000) on gel filtration. The amino acid composition, normalized to 2 residues each of methionine, S-carboxymethyl cysteine and tryptophan, is consistent with the molecular weight determined by gel filtration. Comparison of the amino acid data with the published sequence of the C-terminal portion of the y2-chain [29, 301 indicates that cleavage to form pFc’ occurs in the region of residue number 3 3 1 from the amino terminus (N331) (Table 1). Thus, pFc’ is regarded as an intact C H ~ domain.

-

-

1 13 m

I

0.5

Table 1. Comparison of amino acid compositions of pFc’ and Fc’ with the composition of the C H domain ~ derived from the sequence of y2-chain

0.4

0.3 A280

Icm

Amino acid

pFc’ residues/ moles)

cH3 b)

F c’ residues/ mole

11.3 11 10.2 8.69 9 6.1 12.4 12 9.0 9.21 9 8.0 ClX 10.7 9 8.7 Pro 4.15 4 2.7 GlY I 6.2 Ah 7.1 5 Val 10.8 10 9.0 1.93 2 1.9 Met I le 5.35 6 3.3 6.34 6 6.1 Leu 5.1 1 5 4.9 TYr Phe 3.32 3 3.0 4.00 4 2.5 His 9.48 10 6.8 LY s 4 3.4 Arg 4.22 2.02 2 2.2 SCMCys Trp 2.04 2 ( 2) No. residues 118 116 93.8 MoLwt. 13 424C) 13 088 10 858 13000 t 400d) 11500 ?: 600 A sx

Thr Ser

Residues 338 to 433

0.2

10 6 9

I 8 3 6 9 2 4 6 5 3 3 7 3 2 2 96 10 738

a) From Leslie et al. [18]. b) Residues 331 to C-terminus; compiled from Turner, K.J. and Cebra, J.J. [29j, Trischmann, T.M. and Cebra, J.J. [30]. c) Calculated from amino acid composition. d) Estimated from gel filtration through Sephadex G200 in 5 M guanidinium chloride.

Figure I. Anion exchange chromatographic separation of Fab and Fc products following papain hydrolysis of guinea pig IgG2. Fab p e p tides (PGland 2) eluted with the starting buffer (see Section 3.1.2.), the Fc peptides (Gl-4) were separated by a linear 0 to 0.4 M sodium chloride gradient using an LKB 1130 Ultrograd gradient mixer. The arrow denotes the start of the salt gradient.

Molecular weight estimations o n these peptides (Table 2) were consistent with the approximate molecular sizes assigned under neutral conditions and indicate that Fc‘ behaves as a dimer. The slight difference in molecular weight between cFc and nFc chains, though well within the experimental error, was consistently observed in double labeling experiments employing ‘?-labeled cFc and 1311-labeled nFc in single determinations. Amino terminal sequence studies were performed o n both cFc and n F c after separations by gel filtration in acid and reduction and carboxymethylation with [3H]iodoacetic acid (Table 3). The results indicate that cFc is the product of a single cleavage amino terminal t o the interchain cystine bridge

Eur. J. Immunol. 1976.6: 101-107

M.D. Alexander, R.G.Q. Leslie and S. Cohen

104

Table 2. Physico-chemical properties of Fc fragments from papain hydrolysis of IgGz

Pool n o d GI

+ G2 G3

G4

Relative elution position (Kd) from In PBS In 20 % acetic acid Approx. Pk Relative Kd rno1.wt.b) no. Kd yield mol. w t d ( I ) 0.204

0.268

0.384

5

x 104

3.5 x lo4

2.1 x lo4

Overall molar recovery from digestd)

I

0.104

68

28 2 0 0 * 6 4 0 ( x 2 )

I1

0.342

32

27 8 0 0 k 6 3 0

I I1 111

0.104 0.342 0.567 0.567

60 32

I

Fragment

(%I 14

covalent Fc (cFc) Non covalent Fc (nFc)

6

8 100

27800f630 11 5 0 0 f 6 0 0 11 5 0 0 * 6 0 0

incomplete Fc (iFc) Fc'

28 13 61

a) see Fig. 1. b) Estimated from plot of Kd v. log mol. wt., using BSA (6.7 x lo4 daltons), OA (4.5 x l o 4 ) chymotrypsin (2.5 x lo") and soybean trypsin inhibitor (2.1 x lo4) as references. Vo was determined with dextran blue and Vt with tyrosine. c) Determined from the Kd values of totally reduced and alkylated fragments on Sephadex G-200 in 5 M guanidinium chloride. Estimated from plot of v. 4($'ref. [221). Reference proteins were 72-chains (5.3 x lo4 daltons, L-chains (2.3 x lo4) and lysozyme (1.44 x lo4). All values determined in duplicate except Fc', which was determined in triplicate. d) Assuming AZmTl % = 14 for all fragments.

JKd

Table 3. Amino-terminal sequence analysis of Fc fragments

Fragment

Relative recovery

Sequence

cFc

SCMC Pro Ro Ro Glx Asx

nFc

SCMC Pro Pro Pro Gbr A s Asr Leu Gly Gly (Pro)(Serf Leu Gly Gly (Pro) Ser Val Gly (Pro)(Ser) Val Phe Ile

Gly (Pro) (Ser) Val Phe Ile

0.9 0.1 0.4 0.3

a) Determined by HI hydrolysis of PTH derivatives and amino acid analysis. b) Detected as 3 H after reduction and S-carboxymethylation with [ 3H] iodoacetamide. c) Yields estimated by normalization to PTH norleucine internal standard and correction for losses relative t o norleucine [ 271. Factors for Gly and SCMCys (as Ala) were taken as 1/0.7. d) Turner and Cebra 1291. Arrows indicate sites of papain cleavage.

0.1 0.2

Reported sequenced)

N224

Cvs Pro Lvs . Cvs . Pro Pro Ro Glu A m Leu Gly Gly Pro Ser Val Phe Ile t t t t

Table 4. Carboxyterminal endgroups of Fc fragments performed using hydrazinolysis

Fragment cFca) Fc' Tz-chainb)

The F a b fragments recovered with the pre-gradient lack any class-specific determinants and elute with intact F c on gel filtration in PBS.

C terminal residues (% relative yield) Gly(89) Ser (11) His (30) Gly (19) Leu (19) Ser (16) A h (16) Gly (62) Ser (16) [Asp (8) Ala (7) Leu (7)]

a) Totally reduced and alkylated. b) From Leslie et al. [18].

at N224, while the monomeric polypeptide chains are a mixed population derived from additional cleavages C-terminal to the bridge (caN23 2- 23 5). C-terminal analyses (Table 4) performed by hydrazinolysis show the intact nature of the large F c peptides, contrasting with considerable digestion of the carboxy terminus of Fc', the histidine, leucine and alanine C-termini being consistent with multiple cleavage around residues N43 1433 [30]. The proposed structures for the Fc fragments are shown in Fig. 4 (see discussion).

3.2. Cytophilic activity of native immunoglobulin and its fragments The mean slope value obtained from 9 separate assays of 7 S IgG, was I .89 0.58 x 10-6 for different radio-labeled preparations of 7 S IgG2 of the same antibody specificity. It is considered that this value is comparable with the Ka determined by Leslie and Cohen [20] for 7 S IgG2 of 1.61 f 0.44 x l o 6 l/mole.

*

A total of 6 0 % of the cytophilic activity of IgG2 was recovered in the Fc preparations isolated as gradient pools G I and G 2 from the ion exchange step following papain hydrolysis (Fig. 2 and Table 5 ) . The combined ( G I and G2) pool contained a mixture of cFc and n F c in approximately 2: 1 proportions. The nFc peptides associate under nondenaturing conditions and behave as dimers.

Location of immunoglobulin cytophilic activity

Eur. J. Immunol. 1976.6: 101-107

105

N o cytophilic activity was retained by t h e heterogeneous Fc' fragment isolated as gradient pool G4. Similarly, the pepsinderived pFc' fragment, which incorporates all of the c H 3 domain, retained only 2 5% of the activity of IgG2 (Table 5 and Figs. 2 and 3).

1

4

8

12 16 20 24 28 32 36 Moles X Percentage Maxrrnurn Bmdrng lx105)

44

40

52

48

The fragments with antigen-combining activity, Fab and F(ab')2, demonstrated 3 % or less of the cytophilic activity of IgG2 (Table 5 and Fig. 3). Incomplete purification by immunoadsorption of F(ab')2 preparations gave erroneously high cytophilic activities in some early experiments [20].

3.3. Reduction and alkylation

Figure 2. Inhibition of the binding of 7 S %labeled IgG2 to h o m e logous peritoneal macrophages at 22 "C by the products from papain hydrolysis of guinea pig IgG2. ( - - - - ) Unlabeled 7 S IgG2, (A___ A) cFc/nFc, (BL) iFc, (0-0) Fc', ( 0 0) Fab.

An F c pool isolated by immunoadsorption using a Sepharose 4B-rabbit anti-Faby2 column was treated with varying concentrations of reducing agent in order t o effect reduction of the interchain disulfide bond. The pool contained about 25 % cFc, 5 0 % nFc/iFc and 2 0 5% Fc'. Complete Fc pools generally manifested between 3 7 % and 4 2 5% of the cytophilic activity of IgC2. A rapid drop in cytophilic activity occurred between the aliquot treated with 2 mM DTT and that exposed t o 5 mM D'TT (Table 6). Increasing the reducing agent u p t o 100 mM caused a further slight drop in activity.

120,

Table 6 . Effect or reduction and alkylation on the cytophilic activity of a complete Fc pool 4

8

12

16 20 24 28 32 36 40 Moles x Percentage Maxlmum Binding i X i 0 5 )

44

48

52

Figure 3. Inhibition of the binding of 7 S '251-labeled IgG2 to homologous peritoneal macrophages at 22 "C by the products from peptic 7 S IgG2; ( L A ) hydrolysis of guinea pig IgG.2; (*A) F(ab'h ; O (0)pFc'.

DTT (mM)

Table 5. Cytophilic activity of IgG2 and enzymatically derived fragments Competing ligand IgG2 cFc/nFc iFc F c' Fab F(ab'h pFc'

Range of mb) x

Pool No Expts. no

9 G1 + G 2 4 G3 3

G 4 1 PG1 1 1 3

Meanb) f 1 S.D

Relative binding

(%I - 1.01 to

- 2.80 - 1.89 k0.58 100 1.04 to - 1.29 - 1.13 k0.12 60 - 0.06 to - 0.32 - 0.21 k 0.13 11 0 0 - 0.05 3 - 0.06 3 4-0.1 to - 0.09 - 0.043 f 0.095 2

rb)

Relative binding

(%I 2 5

10 Although antigenically indistinguishable from gradient pools G1 and G2, pool G 3 manifested only about 11 5% of the cytophilic activity of IgG2. This pool contains the iFc fragment which lacks both an interchain disulfide bond and a C H re~ gion from one F C chain, but has dimerized c H 3 domains. Interestingly, the cytophilic activity of this fragment remained consistently above what we consider inactive fragments, e.g. Fab and F(ab'), (Table 5 and Figs. 2 and 3).

ma) x 10-6

100

- 0.70 - 0.41 - 0.14 - 0.14 - 0.04

- 0.95

37

-1 - 0.91

22

- 0.95 - 0.88

7 7 2

a) Slope value (see Table 5) b) r = Correlation coefficient derived using a Monroe 1930 calculator.

3.4. Separation of cFc from nFc and iFc fragments cFc and nFc peptides were separated from one another by G-200 gel filtration in 6 M urea, 0.1 M sodium acetate, pH 4.6. The complete F c pool in 8 M urea was applied t o an upward flow G-200 column in 6 M urea, 0.1 M sodium acetate, pH 4.6. Separated cFc and nFc pools were repeatedly gel filtered t o preclude cross-contamination. Renaturation of urea-treated peptides was achieved by dialysis versus 3 M urea-PBS, 1 M urea-PBS and three t o four changes of PBS at 4 'C.

-

a) See Fig. 1. b) m or slope value derived through linear regression analysis performed using a Monroe 1930 calculator.

Table 7 shows that urea treatment reduced the cytophilic activity of t h e complete Fc pool from 4 2 % to 11 % of the activity of native IgG,. However, 2 8 % of the cytophilic activity was recovered in the pure cFc and 4 % was recovered in the pure nFc pool isolated by gel filtration. Mild reduction of pure cFc dimer with 5 mM DTT reduced the cytophilic activity from 28 % t o 1 0 % of that of IgG,. Similar treatment of the pure nFc raised the activity slightly from 4 % t o 7 %.

106

M.D. Alexander, R.G.Q. Leslie and S . Cohen

Eur. J. Immunol. 1976.6: 101-107

Table 7. Cytophilic activity of cFc and nFc isolated using a dissociating buffer

Domain

CH2

N227

N340

Fc pool Fc pool cFc cFc nFc nFc

-

+ + + + +

DTT (mM)

ma) x

rb)

-

- 0.80 - 0.20

- 0.99 - 0.96

-

- 0.52

5 5

-0.19 - 0.08 - 0.14

- 0.96 - 0.99

Relative binding (%)

-

0.96

- 0.98

c

42 11 28 10 4 7

a) Slope value or m (see Table 5). b) Correlation coefficient (see Table 6).

I I

I

0

I

I

N446

,I

I

Competing Urea ligand

‘H

s-s

s-s

s-s

s-s

s-s

s-s

!

Mol.W1.

4.Discussion The structures of the Fc products of IgG, cleavage, by papain and pepsin, are summarized in Fig. 4. Five chemically distinct species were observed. cFc represents the intact C-terminal portion of IgG, produced by homogeneous cleavage amino terminal t o the inter-chain cystine bridge at N224. nFc is distinguished by additional cleavages C-terminal t o the cystine bridge. Although lacking a covalent link, nFc remains in dimeric form in its native state, as d o the fragments of the c H 3 domain of the ?,-chains ( i e . pFc’ and Fc’: see below); it remains uncertain whether noncovalent interactions occur between CH2 domains. iFc is produced by degradation of one of the paired CH2 domains. Attempts t o recover the free C,2 peptide from the digest mixture have proved unsuccessful, so it is uncertain whether this fragment remains intact, or is progressively degraded. However, the comparative homogeneity in size and net charge of iFc and its dissociation, in acid, into clearly defined monomeric Fc and Fc‘ fragments (Table 2) suggests that inter CH2 - CH3 cleavage with concomitant dissociation of the CH2 domain is an early step in the degradation process. Fc’ and pFc’ are both products of cleavage between t h e CH2 and CH3 domains. They differ in that pFc’ is the product of limited interdomain (peptic) cleavage only, whereas Fc’ displays additional degradation at the carboxy terminal end. The heterogeneous pattern of papain hydrolysis of IgG, appears similar t o t h e hydrolysis of rabbit IgG by papain, though in the latter a variety of hydrolysis conditions were required to produce the different fragments [ 141. The Fab products from papain and peptic cleavage of guinea pig IgG, have been described previously [ 181. However, the precise cleavage points in the ?,-chains giving rise to these fragments remain uncharacterized.

’ of the cytophilic activity of IgG, was recovered About 60 % in F c peptides that were isolated early in t h e ion exchange gradient elution (pools G1 and G2, Fig. 1). Pools G I and G 2 represent a mixture of covalently linked cFc and n F c in a 2: 1 molar ratio (Table 2). Absence of binding activity in the pFc’, Fc’ and both antigen-binding fragments (Table 5) leads us t o conclude that the CH2 domain must play a major part in determining the macrophage cytophilic activity of guinea pig IgG,. The close similarity between the molar activity of the early F c pools (60 %) and the mole fraction of cFc in the pool (0.67) suggests that the covalently linked cFc is responsible for the cytophilic activity. Supporting this contention

23,000

s-s s-s

PFC‘

26.000

II1253.41 Figure 4. Diagrammatic representation of the Fc peptides derived from papain and peptic hydrolyses of guinea pig I&,.

are the studies on purified cFc and nFc, separated in a dissociating buffer. Although urea-acid treatment leads to substantial irreversible loss of activity in the F c pool (Table 6), a clear difference is observed between the cytophilic activity recovered in cFc ( 2 8 %) and nFc ( 4 %). Mild reduction and alkylation of pure cFc with 5 mM DTT caused the cytophilic activity t o fall t o 1 0 % of that of IgG,. Table 5 shows that reduction and alkylation of a native F c pool with 5 or 10 mM DTT substantially reduced the cytophilic activity; interestingly a further reduction in activity occurred with 100 mM DTT. I t seems likely therefore that while the inter heavy chain disulfide bond in cFc is important in maintaining binding activity, a low level of activity may be expressed by the nFc. This conclusion is supported by the residual activity associated with the iFc fragment (1 1 %). Undoubtedly this activity is due in part t o cFc contamination, which in fact amounts to 8 % of the total optical density (Table 2) in the Fc pool, and is equivalent to about 6 % contamination o n a molar basis. The disparity between the observed activity and the measured cFc contamination suggests that even the true monomeric CH2 domain in the iFc may retain low cytophilic activity. Utsumi [ 141 showed that the only papain hydrolysis F c product of rabbit IgG that could sensitize guinea pig skin was a covalently linked Fc (termed 1F c by Utsumi); a fragment lacking the interchain disulfide bond (termed mFc) was inactive, although both fragments retained complement fixing activity. It was concluded that the sites for guinea pig skin fixation were located in the amino terminal part of the 1Fc peptide from rabbit IgG. In the present work, the site(s) on guinea pig IgG, responsible for cytophilic attachment t o the homologous macrophage F c receptor, appear firstly t o be located in the CH2 domain and secondly t o be stabilized by the interchain disulfide bond.

Eur. J. Irnrnunol. 1976.6: 101-107 The apparent location of the macrophage F c receptor binding site in the c H 2 domain of guinea pig IgG, corresponds with the findings of Wisl$ff et al. [ I 11 for another cell binding site; they found that only intact F c and a larger F c fragment, termed Fch, from human IgG3 will effectively inhibit antibody-mediated cytotoxicity, indicating that the CH2 domain or a combination of both CH2 and CH3 was required for the interaction with the cytotoxic cell receptor. In addition, Michaelsen et al. [3 1 ] and Utsumi [ 141 obtained evidence that cellular attachment sites f o r lymphocyte Ig receptors and mast cells, are located in the CH2 domain of rabbit IgG. Ch the other hand, Yasmeen et al. [ 161, Okafor e t al. [ 171 and Ciccimarra et al. [32] located the site for macrophage attachment in the C,3 domain of human IgC (pooled), IgGl and IgG3. In addition Ciccimarra e t al. [32] recently isolated a 10-residue peptide from the C,3 domain of human IgG which inhibits rosette formation with homologous monocytes and binds t o these cells. Unfortunately, the corresponding peptide from the inactive IgG4 subclass which differs by only an Arg for Lys replacement has not been isolated or tested. Ramasamy et al. [33] using mutant IgG secreted by the murine cell line MOPC21 demonstrated that an intact C,3 domain was required for cytophilic attachment of murine IgG MOPC2 1 t o homologous lymphocytes.

It seems unlikely that the absence of cytophilic activity in the isolated Fc’ fragments arises from t h e dependence of the C,3 domain on CH2 for conformational integrity, as has been proposed by Ciccimarra e t al. [32] to explain why human IgGl and IgG3 myeloma proteins with deletions in the C H 2 domains (IgG1 “Dob” and IgG3 “Web”) were less active in inhibiting EA rosettes with homologous monocytes than their intact counterparts. Our observations, those of Michaelsen et al. [31], and Charlwood and Utsumi [34] suggest that stable noncovalent interactions occur mainly between the CH3 domains, since both rabbit pFc‘y2 and guinea pig pFc’y2 behave as noncovalently linked dimers in neutral solution. O n the other hand, rabbit Facb fragments dissociate following mild reduction [3 I ] indicating little interaction between the c H 2 domains. Furthermore full cytophilic activity isdependent, in our system, on a covalent link amino-terminal t o the CH2 domains, and in Michaelsen’s system on the integrity of the C 3 H region. Thus, it seems more likely that conformational stability may be conferred on CH2 by CH3 rather than the reverse. The differences observed in the location of the macrophage Ig receptor binding site may be attributable to the assay techniques used. A macrophage monocyte-erythrocyte rosette test as used by Yasmeen et al. [ 161, Okafor et al. [ 171 and Ciccimarra et al. [32] involves a particulate immune complex whereas the present work assayed the inhibition of celM a r binding of monomeric IgC, by monomeric immunoglobulin fragments. However, the sensitization of macrophage/monocyte monolayers [ 161 was presumably carried out with monomeric immunoglobulin fragments. Differences in receptor sensitivity may occur when the mononuclear cells are bound to glass surfaces. Ciccimara et al. [32] showed that a large proportion of labeled human myeloma proteins were adsorbed by the glass bound macrophage/monocyte monolayers (% 20 % of 10 pg) compared t o the uptake of radiolabeled guinea pig IgG2 by homologous macrophages in suspension monitored in the present work (% 1 % of 2 pg). A systematic comparison of a homologous rosette test with the assay used in the present work is in progress in this laboratory.

Location of immunoglobulin cytophilic activity

107

We would like to thank Hazef Scorts, L.I. BioL, Nicholas Wood, BSc., Laura Smith and Judith Andrews, BSc., f o r their skilled technical assistance. We also wish to express our gratitude to Dr. M.D. Waterfield (ICRF), who carried out the automated sequential cleavage on our behag This research project is supported by the Medical Research Council.

Received September 15, 1975.

5. References 1 Edelman, G.M., Biochemistry 1970. 9 : 31 97.

2 Poljak, R.J., Amzel, L.M., Avey, H.P., Chen, B.L., Phizackerley, R.P. and Saul, F.,Proc. Nat. Acad. Sci. US 1973. 70: 3305. 3 Edelman, G.M., Cunningham, B.A., Gall, W.E., Gttlieb, P.D., Rutishauser, V. and Waxdal, M.J., Proc. Nat. Acad. Sci. US 1969. 63: 78. 4 Wu, T.T. andKabat, E.A., J. Exp. Med. 1970. 132: 211. 5 Reid, K.B.M., Immunology 1971. 20: 649. 6 Sandberg, A.L., Oliveira, B. and Osler, A.. J. Immunol. 1971. 106: 282. 7 Kehoe, J.M., Fougereau, M. and Bourgois, A., Nature 1969. 224: 1212.

8 Kehoe, J.M., Bourgois, A., Capra, J.D. and Fougereau, M., Biochemistry 1974.13: 2499. 9 Ellerson, J.R., Yasmeen, D., Painter, R.H. and Dorrington, K.J., FEBS Lett. 1972.24: 318. 10 Colomb, M. and Porter, R.R., Biochem. J. 1974. 145: 177. 11 Wislbff, F., Michaelsen, T.E. and Froland, S.S., Scand. J. Immunol. 1974.3: 29. 12 Prahl, J.N., Biochem J. 1967. 104: 647. 13 Irimajiri, S., Franklin, E.C. and Frangione, B., Immunochemistry 1968.5: 383. 14 Utsumi, S . , Biochem. J. 1969. 112: 343. 15 Minta, J.O. and Painter, R.H.,fmmunochernistry 1972. 9: 1041. 16 Yasrneen, D., Ellerson, J.R., Dorrington, K.J. and Painter, R.H., J. Immunol. 1973.110: 1706. 17 Okafor, G.O., Turner, M.W. and Hay, F.C., Nature 1974.248: 228. 18 Leslie, R.G.Q., Melamed, M.D. and Cohen, S., Biochem. J. 1971. 121 : 829. 19 Leslie, R.G.Q. and Cohen, S., Immunology 1974. 2 7 : 577. 20 Leslie, R.G.Q. and Cohen, S . , Immunology 1974.27: 589. 21 Michaelsen, T.E. and Nawig, J.B., J. Biol Chem 1974. 249: 2778. 22 Leslie, R.G.Q. and Cohen, S., Biochem. J. 1970.120: 787. 23 Cuatrecasas, P., J. Biol. Chem. 1970.245: 3059. 24 Bradbury, J.H., Nature 1956.178: 912. 25 Fraenkel-Conrat, H. and Tsung, C.M., in Hirs, C.H.W. (Ed.) Methods in Enzymology Academic Press, London and New York 1967.11: 151. 26 Bauer, A.W., Margolies, M.N. and Haber, E., in press. 27 Bridgen, J., Graffeo, A.P., Kargen, B.L. and Waterfield, M.D., in Perham, R. (Ed.) Instrumentation in Amino Acid Sequence Analysis, Academic Press, London and New York, in press. 28 Scatchard, G., N.Y. Acad. Sci 1949.51: 660. 29 Turner, K.J. and Cebra, J.J., Biochemistry 1971. 10: 9. 30 Trischmann, T.M. and Cebra, J.J., Biochemistry 1974. 13: 4804. 31 Michaelsen, T.E., Wislbff, F. and Natvig, J.B., Scand. J. Zmmunol 1975. 4: 71. 32 Ciccimarra, F., Rosen, F.S. and Merler, E., Proc. Nut. Acad. Sci US 1975. 72: 2081. 33 Ramasamy, R., Secher, D.S. and Adetugbo, K., Nature 1975.253: 656. 34 Charlwood, P.A. and Utsumi, S., Biochem. J. 1969. 112: 357.

Cytophilic activity of enzymatically derived fragments of guinea pig IgG2.

Location of immunoglobulin cytophilic activity Eur. J. Immunol. 1976.6: 101-107 M.D. Alexander, R.G.Q. Leslie and S. Cohen Department of Chemical Pat...
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