Immunology 1979 36 487

The binding of rabbit IgG and its enzymatically derived fragments to homologous peritoneal macrophages

MARY GANCZAKOWSKI & R. G. Q. LESLIE Department of Immunology, University Hospital, Queen's Medical Centre, Nottingham

Received 24 April 1978; acceptedfor publication 6 July 1978

1976) and numerous biological studies. The domains involved in antigen binding (VH/VL) (Wu & Kabat, 1970) and fixation of the first component of complement (Cy2) (Kehoe, Fougereau & Bourgois, 1969) have been clearly defined, and activation of the alternative pathway for complement has been tentatively assigned to a third domain (Cy1/CL) (Sandberg, Oliveira & Osler 1971). On the other hand, the domains involved in immunoglobulin attachment to cells are open to dispute. A macrophage binding site in the Cy3 domain ofhuman and murine IgGs has been described in a number of reports (Yasmeen, Ellerson, Dorrington & Painter, 1973, Okafor, Turner & Hay, 1974, Dissanyake & Hay, 1975) and a similar assignment has been made for murine IgG binding to homologous lymphocytes (Ramasamy, Secher & Adetugbo, 1975). By contrast a study of the binding of guinea-pig IgG2 to homologous peritoneal macrophages failed to detect binding activity in an immunoglobulin fragment (pFc') corresponding to the Cy3 domain (Alexander, Leslie & Cohen, 1976). Binding activity was observed with the intact Fc fragment, but direct identification of binding with isolated Cy2 domains proved impossible owing to the lability of this portion of the molecule. In another study employing rabbit IgG and guinea-pig lung macrophages (Ovary et al., 1976), association of binding with the Cy2 domain was established using an IgG fragment (Facb) lacking the Cy3 domian. Criticism may be levelled at this study for its use of a heterologous system which may be detecting a fortuitous interaction between IgG and a cell surface

Summary. Rabbit IgG and its Fab, Fc and pFc' fragments, prepared by papain or peptic digestion, were assayed for binding to homologous peritoneal macrophages. The binding affinity of IgG for the peritoneal macrophages (Ka= 5-9 + 1-6 x 105 L/M) was comparable to that recorded with alveolar macrophages (7-6 ± 1-8 x 105 L/M, Arend & Mannik, 1973) but the number of receptor sites per peritoneal cell (4 6 + 2- 1 x 106) was about four-fold greater than on the latter. Of the fragments, only Fc bound to macrophages with an affinity comparable to intact IgG; pFc' bound weakly and Fab was totally inactive. These data, taken with a recent study involving rabbit IgG and guinea-pig macrophages (Ovary, Saluk, Quijada & Lamm, 1976), indicate that the primary IgG binding site for macrophages is located in the Cy2 domain. INTRODUCTION

The hypothesis that immunoglobulins are comprised of linked, structurally similar units, domains, possessing discrete biological functions (Edelman, 1970) is largely borne out by X-ray crystallographic data (Poljak, Amzel, Avey, Chen, Phizackerley & Saul, 1973, Huber Diesenhofer, Colman, Matsushima & Palm, Correspondence: Dr R. G. Q. Leslie, Department of Immunology, University Hospital, Queen's Medical Centre, Nottingham NG7 2NH. 00 19-2805/79/0300-0487$02.00 © 1979 Blackwell Scientific Publications

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Mary Ganczakowski & R. G. Q. Leslie

component not necessarily involved in binding homologous immunoglobulin. For this reason we decided to examine the binding of rabbit IgG and its fragments to homologous macrophages. MATERIALS AND METHODS

Animals New Zealand white Rabbits (3-5 kg) were used. Preparation ofIgG Rabbit IgG was isolated from pooled sera by precipitation with 40% saturated ammonium sulphate and anion exchange chromatography on DEAE-cellulose (Whatmann, DE32) eluted with 0 021 M Na2HPO4, 0-005 M KH2PO4, pH 7-5 Radio-iodination of IgG was performed as described in Macfarlane (1958). Preparation ofpepsin andpapain hydrolysis products of IgG Peptic digestion of rabbit IgG was performed as described by Mandy, Rivers & Nissonoff (1961) and the pFc' fragment was separated from the other reaction products (F(ab')2 + peptides) by gel filtration through Sephadex GI 50 in phosphate buffered saline (PBS) pH 7-5. Papain digestion (Porter, 1959) yielded a crystalline Fc fragment which was recovered by centrifugation. Fab fragments in the supernatant were contaminated with uncrystallized Fc material which co-eluted with Fab from Sephadex GI 50. Accordingly pure Fab was prepared by papain digestion of the F(ab')2 material from peptic cleavage (see above) followed by passage through GI 50 in PBS Reduction and alkylation of Fc fragments Fc crystals were dissolved in O-5 M Tris-HCI pH 8-5 to give a concentration of 3-4 mg protein per ml. Reduction was carried out with 5, 10 or 100 mM DTT for 45 min at 220 and followed by alkylation with a 10% molar excess of iodoacetamide at 4°. After 30 min, unreacted iodacetamide was removed by dialysis against PBS.

Physical and chemical characterization of the proteolytic fragments Approximate estimates of fragment size in non-dissociating media were made by gel filtration through Sephadex GI 50 in PBS. The column was standardized

with bovine y-globulin (BGG, 150,000 daltons), bovine serum albumin (BSA, 67,000 daltons), ovalbumin (OA, 45,000 daltons), soybean trypsin inhibitor (STI, 21,000 daltons), and ribonuclease A (RNAse, 14,000 daltons), Dextran blue and 1 M NaCl were used to determine the void (V0) and total (Vt) volumes of the column, respectively. Plotting the square root of the elution constant, Kd (=Ve-VO/Vt-V0) for each protein (Ve, elution volume) against the square root of its molecular weight gave a straight line. Non-covalent association of polypeptide chains in the Fc and pFc' fragment was examined by passage through Sephadex G200 in 6 M urea, 0 1 M formic acid, using a column (2-5 x 100 cm, Pharmacia) adapted for upward flow. The column was calibrated with RNAse, STI, OA and BSA which had been totally reduced and alkylated (Leslie, Melamed & Cohen, 1971) prior to loading. Dextran blue and Na125I were used to determine the void and total column volumes. A plot of Kd against mol. wt was used for calibration. Precise measurement of the molecular weights of the polypeptide chains of Fab, Fc and pFc' was made by SDS-polyacrylamide gel electrophoresis using a horizontal thin-layer gel of 10% acrylamide, 0-27% N,Nmethylene-bis-acrylamide in 1% SDS, 0-01 M phosphate buffer pH 71, as described by Fehrnstrom & Moberg (1977). BSA, OA, carbonic anhydrase (CA, 29,000 daltons), myoglobin (MYo, 17,200 daltons) RNAse and cytochrome C (Cyt C, 12,500 daltons) were employed as standards. The relative mobility (w.r.t. bromophenol blue) of each protein was plotted against logl0 of its molecular weight. Linear regression analyses were performed with a Compucorp 325 desk top computer. Amino acid analyses of Fc and pFc' fragments were performed on a Locarte amino acid analyser as described previously (Leslie & Cohen, 1970). The tryptophan content of pFc' was determined spectrophotometrically (Melamed & Green, 1963). Cell preparation Rabbit peritoneal exudate cells were harvested 4 days after stimulation by injection of sterilized liquid paraffin as described for guinea-pigs (Leslie & Cohen, 1974). The yield of cells ranged between 2 5 and 10 x 107 per rabbit, containing, on average, 74% macrophages (range = 64-82%), 20% lymphocytes and 6% polymorphonuclear leucocytes. The cells were washed twice with TC199 (20 ml), with incubation for 15 min at 370, before assay.

489

Binding ofIgG andfragments to peritoneal macrophages

Macrophage binding assay Before assay, rabbit IgG and fragments were passed through Sephadex GI 50 in PBS and spun at 200,000g for 90 min to remove aggregates over 21S in size. The binding assay has been described in detail in earlier publications from this laboratory (Leslie & Cohen 1974; Alexander et al., 1976). Essentially 1251I-labelled IgG (2pg) in 1 ml TC199 containing 1% OA was incubated with peritoneal exudate cells (2 x 106) at 22° for 90 min in the presence of unlabelled IgG or fragment (5-200pg). The cells were washed and cell bound counts determined. The binding affinity of IgG was determined by plotting (ratio of bound IgG: free IgG) against (concentration of bound IgG) (Scatchard, 1949). In the fragment studies, the data are normalized, for the purpose of comparison, by expressing 1251I-IgG uptake in the presence of inhibitor fragment (or IgG) as a percentage of the calculated uptake of 125 I-IgG at infinite dilution (see Alexander et al., 1976). RESULTS

Characterization of Rabbit IgG and its fragments IgG. Purified rabbit IgG produced a single arc on immunoelectrophoresis against a goat antiserum to rabbit serum, and eluted as a single, symmetrical peak from Sephadex GI 50 in PBS. Fragments from pepsin digestion. Gel filtration on Sephadex GI 50 in PBS resolved the pepsin hydrolysate of rabbit IgG into three peaks; an F(ab')2 peak with a molecular weight of about 110,000 daltons, a pFc' peak (about 21,000 daltons) and small peptides. F(ab')2 and pFc' fragments were antigenically distinct and deficient with respect to whole IgG. pFc' displayed an apparent molecular weight of 12,400 on passage through Sephadex GI 50 in 6 M urea, 0-1 M formic acid indicating that in its native state, the fragment exists as a non-covalently linked dimer (Table 1). After reduction in 1% SDS, F(ab')2 ran as two bands on polyacrylamide gel electrophoresis, with molecular weights of 25,000 + 580 daltons (L-chain) and 28,100 + 600 daltons (H-chain fragment), while pFc' ran as a single band with molecular weight of 11,900 + 400 (Table 1). The amino acid composition of pFc' when normalized to three residues of methionine per polypeptide chain, is consistent with the molecular weight determined by SDS-PAGE (Table 2). Comparison of the amino acid data with the published sequence of the

Table 1. Size estimations on fragments employed in the binding studies

Sephadex GI 50 elution in PBS Fragment

Kd

Fab Fc

0 33 0 30

pFc' IgG

F(ab')2

Sephadex G200 elution in 6 M urea

Approx. SDS-PAGE Approx. mol. wt Kd mol. wt mol. wt. + 1 SD

45,000 50,000 0-17 0-36 049 21,000 052 0-030 166,000 0-17 0-36 0-11 110,000 -

-

53,000 25,000 12,400 53,800 24,900 -

25,020+ 580 25,700+490 23,400+ 310 11,920+400 54,149+200 25,000+600 25,000+600 28,050+610

C-terminal portion of rabbit y chain (Hill, Delahey, Lebovitz & Fellows 1966) confirms that the pFc' fragment corresponds to the Cy3 domain. Fragmentsfrom papain digestion. Papain cleaves rabbit IgG to yield an Fc fragment which crystallizes from low ionic strength buffer (Porter, 1959) and is antigenically dificient with respect to whole IgG, though less so than pFc', with which it shares determinants. In neutral buffer, it behaves as two components on Sephadex G150; a minor aggregated component eluting with the void volume of the column and a major symmetrical peak about 50,000 daltons in size. On gel filtration in 6 M urea, 0-1 M formic acid (in the presence of 50 mm iodoacetamide, to prevent disulphide rearrangement) Fc resolved into two peaks in a ratio of 1: 15 (Fig. 1) with approximate molecular weights of 53,000 and 25,3000 respectively; an indication that the Fc is present both in an intact disulphide bridged form (cFc, Fig. 2) and as a non-covalent dimer of j y-chains (nFc) resulting from papain cleavage C-terminal to the inter-heavy chain disulphide bridge. After reduction in SDS, the Fc fragment resolved into two bands with molecular weights of 25,700 + 490 and 23,400 + 300, reflecting the variation in cleavage points indicated by the partial dissociation in urea. The Fab fragment which remained in solution at low ionic strength was still contaminated with material carrying Fc determinants, so pure Fab was prepared by papain hydrolysis of the F(ab')2 fragment from peptic digestion. SDS-PAGE failed to resolve reduced Fab into two bands indicating that the H-chain fragment has a molecular weight close to that of L-chain (25,000 + 600 daltons).

490

Mary Ganczakowski & R. G. Q. Leslie Table 2. comparison of the amino acid composition of pFc' with the sequence-determined composition of the Cy3 domain. pFc' Residues*/mole

Amino acid

Asp Thr Ser Glu Pro Gly Ala Val

Cy3 Residues in sequence N339-C terminus

3 11 6 29 492 1 82 107 52

9 7 15 11 8 7 4 7 2 3 3 7 6 3 3 6 5 2 108

12,192

12,241

905 6 91 13 01 10-83 7.37 7 61

4*35 8 14 1-84 3 00 3 06 681 6 19 3 23

jCys Met Ile Leu Tyr Phe His Lys Arg Trp Total no. of residues Mol. wt, from AA composition Mol. wt determined by SDS-PAGE

11,900 + 4000

* Determined by duplicate amino acid analyses and normalized to 3 moles methionine per mole of pFc'. t From the reported sequence (Hill et al., 1966). Residue numbering has been taken from the guinea-pig y2-chain.

Domain

N230

I

20,000

C73

Cy2

N446

N339

I71

Mol. wt. 51,400 (covalent)

(covalent)Fc

--7-S-S-, 15,000 C!

dI10,0001

tIWjjH

5,0001

0

30

40

50

60

70

80

-Y~S-S-~ ~ Fc (non- 47,000to 7-S-S v

covalent) 51,000

pFc

23,800

Fraction No

Figure 1. Gel filtration of rabbit Fc through Sephadex G200 in 6 M urea, 0-1 M formic acid in the presence of 50 mm iodoacetamide.

Figure 2. Schematic representation of the Fc region fragments produced by papain and pepsin digestion of rabbit IgG.

Binding ofIgG andfragments to peritoneal macrophages Macrophage binding activity of IgG and its fragments IgG. In six separate assays IgG bound with uniform affinity to peritoneal exudate cells as judged by the linear Scatchard plots obtained with correlation coefficients, r, of between -0 80 and -1 00 (mean r,-0 92). The mean affinity was 586 + 1 56 x l05 L/M with an average of 4 6 +21 x 106 IgG receptor sites per macrophage.

Fab, Fc andpFc'fragments. Of the fragments tested, only Fc displayed substantial capacity to inhibit the binding of 1251-IgG (Fig. 3). In four experiments, the mean binding activity of Fc, relative to IgG was 119% (Table 3). The slightly raised level of binding activity was observed in spite of attempts to remove aggregates by gel filtration and ultra-centrifugation. +

A

491

pFc' displayed a low level of inhibitory activity in two out of three experiments and slight enhancement in the third. Correlation coefficients in all three experiments were low (r= -0 51,-0 34 and 0-68). A mean relative binding activity of 7% was recorded with a standard deviation of similar magnitude indicating that the activity cannot be regarded as significant (Table 3). The Fab fragment was similarly inactive in a single assay. Table 3. Binding activity of rabbit Ig fragments for peritoneal macrophages

Relative Competing ligand

No. of expts

IgG Fab pFc' Fc

6 1 3 4

+

1I

Mean apparent

K. (L/M

x

I0-i)

5 86 + 1-56 -0-32

039+0-32 697+095

binding (%) 100 -5 7 119

0'

0~

0

I I I

I.

40 80 [Inhibitor] xmaximum binding

I

I 120

\j

160

(Mx1010)(%)

Figure 3. Inhibition of the binding of 7S 1251-labelled IgG to homologous peritoneal macrophages at 220 by IgG ( and its Fc (*), Fab ( + +) and pFc' (A-----) fragments.

Effect of disulphide cleavage on Fc binding activity The quantitative recovery of binding activity in an Fc pool containing both intact Fc fragments (cFc) and non-covalently linked I y-chain dimers (nFc) indicated that integrity of the inter-y- chain disulphide bridge was unimportant in maintaining cytophilic activity. To test this, Fc was reduced with three concentrations of DTT and tested for residual binding activity. Low concentrations of DTT (5 and 10 mM) were without significant effect on the cytophilic activity of Fc (Table 4) while at 100 mm DTT a substantial drop in binding activity was recorded.

Table 4. Effect of reduction and alkylation on rabbit Fc binding to macrophages DTT

Expt 1 Expt 2

(mM)

Apparent Ka (L/M x 10-5)

r

Relative binding

0 5 10 0 100

5 55 4-52 6-29 7-38 3 10

-0-85 -0-72 -0-74 -0 79 -0-63

100 82 114 100 42

Mary Ganczakowski & R. G. Q. Leslie

492

DISCUSSION

The structures of the Fc products of IgG cleavage by papain and pepsin are shown in Fig. 2. Correspondence of the pFc' fragment with the Cy3 domain has been confirmed from its molecular weight and the near identity of its amino acid composition with that derived from the reported sequence (Hill et al., 1966). The crystalline Fc fragment is a mixture of two forms (cFc and nFc) which arise from papain cleavage at two or more positions in the region of the inter-chain disulphide bridge. In molecular weight determinations on this mixture, two polypeptide chains of 25,700 + 490 daltons and 23,400 + 300 daltons were identified. Allowing 1,500 daltons for carbohydrate (3% of an intact y-chain), the molecular weight of the larger fragment is still great enough to include at least the inter-chain cystine bridge which lies 217 residues from the carboxy terminus (O'Donnel, Fragione & Porter, 1970) whereas the smaller chain is produced by cleavage about twenty residues C-terminal to the cystine. Precise comparison of the amino acid composition of Fc with the reported sequence (O'Donnell et al., 1970; Fruchter, Jackson, More & Porter, 1970, Hill et al., 1966) is not possible because of the heterogeneity arising from multiple cleavage points. Overall similarities between the two are apparent, however (data not shown). The heterogeneity of cleavage recorded here is consistent with that described by Utsumi (1969) for papain digestion of rabbit IgG under reducing conditions and is comparable to the proteolysis observed with guinea-pig IgG2 (Alexander et al., 1976). Intact rabbit IgG binds at 220 to peritoneal macrophages with an affinity (5 86 + 1-56 x 105 L/M) comparable to that reported at 370 with alveolar macrophages (7-59+1 83x 105 L/M, Arend & Mannik, 1973). On the other hand the number of receptor sites per peritoneal macrophage (4 6 + 2 1 x 106 sites/cell) is about four times the reported figure for minimally stimulated alveolar macrophages (1 -21+ 0-23 x 106 sites/cell) and twice that for heavily stimulated lung macrophage (2 16 + 0-34 x 106 sites). Presumably this variation reflects the differences in cell source as well as in the degree of stimulation. The present study establishes that the binding activity of rabbit IgG to homologous peritoneal macrophages is retained in the whole Fc fragment, which contains Cy2 and Cy3 domains but not by pFc', consisting of Cy3 domains alone. In this respect, rabbit IgG is similar in its activity to guinea-pig IgG2 (Alexander et al., 1976) On the other hand, the two species

differ markedly in their requirements for covalent integrity within the Fc region. In guinea-pigs, full binding activity was associated only with the cFc fragment and not nFc whereas the rabbit Fc pool binds to macrophages as effectively as intact IgG in spite of the high proportion of nFc present. The two species of Fc also differ markedly in their susceptibility to reduction. Low concentrations of dithiothreitol (2-10 mM), sufficient to cleave inter-chain disulphide bonds only, lead to virtual abolition of guinea-pig Fc cytophilic activity, but have little effect on the function of rabbit Fc (Table 4). In the case of the guinea-pig it was concluded that the Cy2 domains required constraint both at the amino-terminal end (by cystine bridging) and at the carboxy-terminal end (through non-covalent association of the Cy3 domains) to maintain its native conformation and functional integrity. Similar observations have been made for rabbit IgG as a mediator of human K-cell activity (Michaelsen, Wisl0ff & Natvig, 1975) and of histamine release from guinea-pig mast cells (Utsumi, 1969). The present data suggest no such requirement for the binding of rabbit IgG to macrophages, though the activity does appear to depend on the integrity of intra-chain bridges as judged by the effectiveness of 100 mM DTT in reducing Fc function (Table 4). The stability of the macrophage-binding activity of rabbit IgG is comparable to the immunoglobulin's ability to fix complement (Utsumi, 1969). Thus one might expect that, as in complement fixation, free Cy2 domains (Yasmeen, Ellerson, Dorrington & Painter, 1976) or the Facb fragment (MacLennan, Connel & Gotch, 1974; Colomb & Porter 1975) would retain detectable binding activity, Ovary et al., (1976) have employed this approach in the study of rabbit IgG binding to guinea-pig lung macrophages using a rosette assay. They demonstrated that erythrocytes sensitized with either IgG or Facb would form rosettes with the macrophages which could be inhibited with free IgG or Fc fragment but not pFc'. The objection that the use of a heterologous system might have lead to binding artefacts is countered by the following observations: (1) rabbit IgG (unlike human IgGI or IgG3) binds to guinea-pig macrophages with uniform affinity to the same receptor sites as guinea-pig IgG2 (R.G.Q. Leslie and A. Niemetz, unpublished observations) indicating that rabbit IgG is cross-reacting with an Fc receptor on guinea-pig macrophages which is probably similar in structure to the receptor on homologous cells; and (2) the relative inhibitory activities of rabbit Fc and pFc', with respect to whole IgG, is the

Binding ofIgG andfragments to peritoneal macrophages same in both heterologous and homologous systems which suggests that the same binding site on rabbit IgG is involved in both cases. The evidence in both guinea-pigs and rabbits clearly indicates involvement of the Cy2 domain in IgG binding to macrophages. In mice and humans the data are conflicting, with a majority of reports indicating involvement of the Cy3 domain (Yasmeen et al., 1973; Okafor et al., 1974; Dissanayake & Hay, 1975; Ciccimara, Rosen & Merler, 1975). Detailed discussions of these differences have appeared in recent publications from this laboratory (Alexander et al., 1976; Alexander, Andrews, Leslie & Wood, 1978) and elsewhere (Ovary et al., 1976). In summary, the discrepancy in findings has been attributed to: (1) differences in assay techniques (Alexander et al., 1976); (2) differences between systems studied in terms ofanimal species and types of macrophage or monocyte used (Leslie, Alexander & Cohen, 1976; Ovary et al., 1976); and (3) the existence of multiple binding sites or co-operating regions in Cy2 and Cy3 domains (Ovary et al., 1976). In a recent publication (Alexander et al., 1978), the two major techniques employed (radioassay and rosette formation) have been compared and shown to give consistent results. In the same study, human and guinea-pig systems were compared and found to be similar in their lack of Cy3 activity. The possibility of domain co-operation remains, though the demonstration that Facb is as effective, quantitatively, as whole IgG in rosette formation (Ovary et al., 1976) argues against this mechanism. Recent studies of IgG binding to placental and gut epithelial tissue (McNabb, Koh & Painter, 1976, Guyer, Koshland & Knopf, 1976) suggest that cell attachment via the Cy2 domain may be a general phenomenon. In the face of contradictory evidence (see above), however, further experimentation is required to test the validity of this view.

ACKNOWLEDGMENTS The authors are indebted to Andy Macpherson and Frank Serebour for their skilled assistance in performance of amino acid analyses and molecular weight determinations. We would also like to thank Dr M. D. Alexander and Professor Sidney Cohen for their valuable criticism and encouragement.

REFERENCES ALEXANDER M.D., ANDREWS J.A., LESLIE R.G.Q. & WOOD

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N.W. (1978) The binding of human and guinea-pig IgG subclasses to homologous macrophage and monocyte receptors. Immunology, 35, 115. ALEXANDER M.D., LESLIE R.G.Q. & COHEN S. (1976) Cytophilic activity of enzymatically derived fragments of guinea-pig IgG2. Europ. J. Immunol. 6, 101. AREND W.P. & MANNIK M. (1973) The macrophage receptor for IgG: number and affinity of binding sites. J. Immunol. 110, 1455. CICCIMARA F., ROSEN F.S. & MERLER E. (1975) Localisation of the IgG effector site for monocyte receptors. Proc. nat. Acad. Sci. U.S.A. 72, 2081. COLOMB M. & PORTER R.R. (1975) Characterisation of a plasmin digest fragment of rabbit immunoglobulin gamma that binds antigen and complement. Biochem. J. 145, 177. DISSANYAKE S. & HAY F.C. (1975) Investigation of the binding site of mouse IgG subclasses to homologous peritoneal macrophages. Immunology, 29, 111. EDELMAN G.M. (1970) The covalent structure of a human yG-immunoglobulin. XI. Functional implications. Biochemistry, 9, 3197. FEHRNSTROM H. & MOBERG U. (1977) SDS and conventional polyacrylamide gel electrophoresis with LKB 2117 Multiphor. L.K.B. Application note 306. FRUCHTER R.G., JACKSON S.A., MOLE L.E. & PORTER R.R. (1970) Sequence studies of the Fd section of the heavy chain of rabbit IgG. Biochem. J. 116, 249. GuYER R.L., KOSHLAND M.E. & KNOPF P.M. (1976) Immunoglobulin binding by mouse intestinal epithelial cell receptors. J. Immunol. 117, 587. HILL R.L., DELANEY R., LEBOvITZ H.E. & FELLOWS R.E. JR. (1966) Studies on the amino acid sequence of heavychains from rabbit immunoglobulin G. Proc. Roy. Soc. B. 166, 159. HUBER R., DIESENHOFER J., COLMAN P.M., MATSUSHIMA M. & PALM W. (1976) Crystallographic structure studies of an IgG molecule and an Fc fragment. Nature (Lond.), 264,415. KEHOE J.M., FOUGEREAU M. & BOuRGoIS A. (1969) Immunoglobulin peptide with complement fixing activity. Nature (Lond.), 224, 1212. LESLIE R.G.Q., ALEXANDER M.D. & COHEN S. (1976)

Bindingofimmunecomplexesofguinea-pigIgG2tohomologous peritoneal exudate cells. Europ. J. Immunol. 6,841. LESLIE R.G.Q. & COHEN S. (1970) Chemical properties of guinea-pig immunoglobulin yIG, y2G and yM. Biochem. J. 120, 787. LESLIE R.G.Q. & COHEN S. (1974) Cytophilic activity of IgG2 from sera of unimmunised guinea-pigs. Immunology, 27, 577. LESLIE R.G.Q., MELAMED M.D. & COHEN S. (1971) The products from papain and pepsin hydrolyses ofguinea pig immunoglobulins y1G and Y2G. Biochem. J. 121, 829. MAcFARLANE A.S. (1958) Efficient trace labelling of proteins with iodine. Nature (Lond.), 182, 53. MACLENNAN I.C.M., CONNEL G.E. & GOTCH F.M. (1974) Effector activating determinants on IgG. II. Differentiation of combining sites for Clq from those for cytotoxic K-cells and neutrophils by plasmin digestion of IgG. Immunology, 26, 303. MACNABB T., KOH T.Y. & PAINTE R.H. (1976) Structure

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Mary Ganczakowski & R. G. Q. Leslie

and function of immunoglobulin domains.V.Binding of IgG and fragments to placental membrane preparations J. Immunol. 117, 882. MANDY W.J., RIVERS M.N. & NISONoFF A. (1961) Recombination of univalent subunits derived from rabbit antibody. J. biol. Chem. 236, 3221. MELAMED M.D. & GREEN N.M. (1963) Avidin 2. Purification and composition. Biochem. J. 89, 591. MICHAELSEN. T.E., WISLoFF F. & NATVIG J.B. (1975) Structural requirements in the Fc region of IgG antibodies necessary to induce cytotoxicity, by human lymphocytes. Scand. J. Immunol. 4, 71. O' DONNELL I.J., FRANGIONE B. & PORTER R.R. (1970) The disulphide bonds of the heavy chain of rabbit IgG Biochem. J. 116,261. OKAFOR G.O., TURNER M.W. & HAY F.C. (1974) Location of monocyte-binding site of human immunoglobulin G. Nature (Lond.), 248, 228. OVARY Z., SALUK P.H., QUIJADA L. & LAMM M.E. (1976) Biological activities of rabbit immunoglobulin G in relation to domains of the Fc region. J. Immunol. 116, 1265. POLJAK R.J., AMZEL L.M., AVEY A.P., CHEN B.L., PHIZACKERLEY R.P. & SAUL F. (1973) Three dimensional structure of the Fab' fragment of a human immunoglobulin at 2-8 A resolution. Pro. nat. Acad. Sci. U.S.A. 70, 3305. PORTER R.R. (1959) Hydrolysis of rabbit y-globulin and

antibodies with crystalline papain. Biochem. J. 73, 119. RAMASAMY R., SECHER D.S. & ADETUGBO R. (1975) CH3 domain of IgG as binding site Fc receptor on mouse lymphocytes. Nature (Lond.) 253, 656. SANDBERG A.L., OLIVEIRA B. & OSLER A. (1971) Two complement interaction sites in guinea pig immunoglobulins. J. Immunol. 106, 282. SCATCHARD G. (1949) The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660. UTSUMI S. (1969) Stepwise cleavage of rabbit IgG by papain and isolation of four types of biologically active Fc fragments. Biochem. J. 112, 343. Wu T.T. & KABAT E.A. (1970) An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. exp. Med. 132, 21 1. YASMEEN D., ELLERSON J.R. DORRINGTON K.J. & PAINTER R.H. (1973) Evidence for the domain hypothesis: location of the site of cytophilic activity towards guinea pig -macrophages in the CH3 homology region of human immunoglobulin G. J. Immunol. 110, 706. YASMEEN D., ELLERSON J.R., DORRINGTON K.J. & PAINTER R.H. (1976) The structure and function of immunoglobulin domains. IV. The distribution of some effector functions among the Cy2 and Cy3 homology regions of human immunoglobulins G. J. Immunol. 116, 518.