Proteins separated from human IgG molecules

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Molecular Immunology, Vol. 30, No. 10, pp. 935-940, 1993 Printed

Pergamon Press Ltd

in Great Britain.

PROTEINS

SEPARATED

FROM HUMAN

IgG MOLECULES

ROALD NEZLIN,* ANDREW FREYWALD and MARTIN OPPERMANN~ Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot I-76100, Israel and TDepartment of Immunology, University of Gijttingen, Gdttingen D-3400, Germany (Received

13 December 1992; accepted 17 January 1993)

Abstract-Immunoglobulin G binding proteins were separated from human IgG molecules using 1 N acetic acid followed by 5 M guanidinium chloride in 0.1 M acetic acid. The proteins thus obtained were heterogeneous as demonstrated by SDS-PAGE and reverse-phase HPLC. The isolated proteins consisted of two types: the C3a and C4a complement fragments (anaphylatoxins) and immunoglobulin peptide chain fragments V,I and C,3. Both anaphylatoxins immobilized on cellulose nitrate membranes could reassociate with intact IgG molecules. The ubiquitous presence of C3a in IgG preparations was demonstrated using monoclonal antibodies specific for C3a. Nearly all of the bound anaphylatoxin molecules were found in the Fab fragment. These findings suggest that IgG molecules can eliminate anaphylatoxins from the circulation, and thus prevent harmful effects due to these active complement components.

identified terization

INTRODUCTION Immunoglobulin domains are well suited for the construction of protein units that can react with different ligands. Members of the immunoglobulin superfamily that are composed of these domains are well known for their recognition capacity. Beside recognizing antigens by their antigen combining sites, immunoglobulins can also bind protein ligands by their constant domains. Two well known examples are the binding of the complement component Clq and the staphylococcal protein A and G by the Fc portions of IgG molecules. Recently, using strong dissociating denaturants, we separated a heterogeneous group of IgG binding proteins (IBP) from human IgG. Among these IBP were anaphylatoxins-the complement components C3a and C4a (Nezlin and Freywald, 1992). The anaphylatoxins C3a and C4a are small proteolytic fragments consisting of 77 amino acid residues. They are released from the C3 and C4 complement components, respectively, upon activation of the complement cascade. Circulating cells as well as many tissue cells including mast cells and macrophages respond to these factors. Anaphylatoxins can activate various secondary mediator systems including vasoamines, prostaglandins and leukotriens. These mediators enhance vascular permeability and induce contraction of smooth muscles (for review see Vogt, 1986; Hugli, 1989). C3a is also immunosuppressive. In addition to anaphylatoxins, two fragments of immunoglobulin peptide chains (V,I and C,3) were

among IBP. Herein the isolation and characof both groups of IBP are described. MATERIALS

AND METHODS

Reagents Acetotonitrile HPLC grade was obtained from Bio-Lab (Jerusalem, Israel) and trifluoroacetic acid (TFA) was from Merck (Darmstadt, Germany). Guanidinium hydrochloride (GuHCl), tris(hydroxymethy1) aminomethane, Tween 20, papain (2 x , crystallized, lyophilized powder), ovalbumin, bovine and human serum albumins were purchased from Sigma (St. Louis, MO). Iodobeads (Pierce, Rockford, IL); DEAE 32cellulose (Serva, Heidelberg, Germany); cellulose nitrate membranes BAS 85 (Schleicher & Schuell, Dassel, Germany); and Immobilon-P transfer membranes (Millipore, Bedford, MA) were obtained as noted. Protein preparations Immunoglobulin G (IgG) was isolated from the plasma of healthy donors or patients with multiple sclerosis by precipitation with ammonium sulphate at 45% saturation followed by DEAE ion exchange chromatography in 0.005 M phosphate buffer, pH 8.5. IBP were separated from IgG by membrane filtration of IgG solutions in an Amicon cell with 50 K membranes as follows. Lyophilized IgG (2-4 g) was dissolved in phosphate buffered saline (PBS, 200 ml) and concentrated to 25 ml in the Amicon cell. The 25 ml of IgG solution in the cell were diluted to 200 ml with PBS and concentrated to 25 ml again. Acetic acid (1 N, 180 ml) was then added to the concentrated IgG solution (25 ml) in the Amicon cell and the mixture was concentrated to 25 ml. The resulting filtrate (the acetic acid filtrate) was collected. Finally, 180 ml of 6 M GuHCl was added to the concentrated IgG solution in acetic acid and the mixture

*Author to whom correspondence should be addressed. IBP, immunoglobulin G binding proteins; mAbs, monoclonal antibodies; PBS, phosphate buffered saline; HPLC, high pressure liquid chromatography; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Abbreviations:

935

936

ROALD NEZLIN et al.

was concentrated to 25 ml. The resulting filtrate (GuHCl filtrate) was collected. Acetic acid and GuHCl were removed from the filtrates by repeated membrane filtrations in Amicon cells using 3 K membranes or by dialysis against PBS. Fab and Fc fragments were isolated from papain hydrolysates (digestion time 4 hr at pH 7.5) of IgG by DEAE ion exchange chromatography. Fab fragments were eluted with the starting buffer (0.005 M, pH 8.0) and Fc fragments-by starting buffer containing 0. I5 M NaCl. The preparations of Fab and Fe fragments were both passed through a Sephadex G-200 column to separate the fragments from undigested IgG molecules and aggregates.

The monoclonal antibodies (mAbs) Dl7/1 and Kl3/16, generated from BALB/c mice immunized with purified human C3a, are both IgGl/rc (Oppermann et al., 1987). They recognize C3a(desArg), and do not crossreact with human C4a or C5a. Dl7/1 and Kl3/16 recognize different epitopes on the 9 kDa C3a (Oppermann et al., 1988). Kl3/16 effectively inhibits the biological activity of C3a (Piische et al., 1989) whereas 017/l does not. Neither mAb reacts with a synthetic octapeptide representing the C3a-carboxyterminus (Oppermann, unpublished). SDS-PAGE

electrophoresis

Linear gradient SDS-gels (8-22% acrylamide) with high concns of tris(hydroxymethyl)aminomethane in the resolving gel (0.75 M) and running buffer (0.05 M) (Fling and Gregerson, 1986) or 12% gels according to the Laemmli system (Gallaher and Smith, 1991) were used for protein analysis. Gels were stained by Coomassie Brilliant Blue G or silver staining, according to the Bio-Rad manual. High Pressure liquid Chromatography (HPLC) IBP were fractionated by reverse-phase HPLC on a Hewlett Packard Chemstation 7999414 equipped with a HP 1040A Diode-Array Detector and a HP pump 1050. The Brownlee RP-300, Aquapore Octyl reverse phase columns (30 x 4.6 mm) were purchased from Applied Biosystems, Inc. (Foster City, CA). Dried samples of the proteins to be analyzed were denatured by adding 6 M GUI-ICI in 0.1% TFA. The column was equilibrated with 0.1% TFA (95% pump A) and 0.085% TFA/SO% acetonitrile (5% pump B) mixture. The sample volume did not exceed 100 ~1 per injection. The following gradient program was used for separation (Matsudaira, 1990): O-5 min, 5% B; 5-45 min, 70% B; 45-50 min, 100% B; 50-55 min, 5% B; flow rate 0.5 ml/min. Peaks were collected manually and concentrated in a Speed-Vat centrifuge. Amino acid sequencing IBP separated by reverse phase HPLC or by SDS-PAGE and immunoblotting were subjected to amino acid sequencing. IBP-containing HPLC fractions were concentrated to approximately 15 ~1 in a Speed-

Vat centrifuge. Alternatively IBP were subjected to SDS-PAGE and blotted onto Immobilon P (polyvinylidene difluoride-PVDF) membranes in 5OmM HEPES buffer (pH 7.5) containing 20% methanol, at 300 mA for 3-6 hr in cold (Matsudaira, 1987). The membrane was then washed with water, stained with Coomassie blue (0.25% in 50% methanol and 7% acetic acid) and excess dye was removed by 50% methanol in 10% acetic acid. The stained bands were cut out after drying and subjected to sequencing by an Applied Biosystems 475 A sequencer. Dot blots Protein solutions (5- 10 ~1) were applied to pieces of cellulose nitrate membranes. After one hour at room temp the membranes were washed three times with PBS containing 0.2% Tween 20 as a blocking agent. The immobilized proteins were then incubated at room temp with “51-labeled proteins or antibodies for one hour. After washing with PBS. the radioactivity of the membranes was determined by a gamma-mounter, or autoradiography using AGFA Curix RP-2 films. The developed films were counted on a computing densitometer (Model 300A, Molecular Dynamics, Sunnyvale, CA).

rz51labeling was achieved by the chloramine method using Iodo-beads according to the procedure described in the Pierce Catalog and Handbook (Rockford, IL). RESULTS Fractionation ofimmunoglobuiin G binding proteins Electrophoresis of isolated IgG preparations revealed bands corresponding to the H and L chains of IgG and other bands. The additional bands were usually faint and clearly visible only upon overloading of gels. The bands could be due to proteins that copurify with IgG or whose binding affinity for IgG molecules is significant. Proteins that copurify with IgG were removed by the membrane filtration in PBS. Immunoglobulin G binding proteins (IBP) were separated from IgG by membrane filtration using 1 N acetic acid followed by 5 M GuHCl in 0.1 N acetic acid. Typically about 2 mg of IBP were isolated from 1 g of freeze-dried human IgG preparations. Fractionation of IBP by SDS-PAGE revealed several bands, whose apparent molecular masses correspond to about 25 kDa and 8-14 kDa (Fig. 1). The number and position of the bands were different in the acetic acid and GuHCl filtrates. Fractionation of IBP by reverse-phase HPLC revealed three main groups of protein molecules. One, of gradient which appeared at the beginning (13-17 min), was present only in the acetic acid filtrates (Fig. 2), while the other two, which appeared later, were present in both the acetic acid and GuHCl filtrates (Fig. 3A). SDS-PAGE revealed that the proteins in the early peak (fractions l-3 of the acetic acid filtrates, Fig. 2)

Proteins

separated

from human

IgG molecules

336

-

937

-

66

29 f 24 -20 -

14

Fig. 1. SDS-PAGE of proteins separated from human IgG by 1 N acetic acid alone (two left lanes) or followed by 5 M GuHCl in 0.1 N acetic acid (two right lanes). The positions of the molecular mass markers (kDa) are indicated on the left. Two linear gradient acrylamide gels (S-22%) with high concns of tris(hydroxymethyl)aminomethane in the resolving gel (0.75 M) and in the running buffer (0.05 M) were used.

240

7

9

II

13

15

17

Minutes

Fig. 2. Reverse-phase HPLC fractionation of proteins separated from human IgG by 1 N acetic acid (only the first main peak is presented).

-

I

I

I

I

25

30

35

40

Minutes

36 29

20 14 6

Fig. 3. (A) Reverse-phase HPLC fractionation of proteins separated from human IgG by 5 M GuHCl in 0.1 M acetic acid (after treatment with 1 N acetic acid). (B) SDS-PAGE analysis of the proteins of peaks 1-7 (Fig. 3A). See legend to Fig. 1 for conditions. The positions of the molecular mass markers (kDa) are indicated on the left.

938

ROALD NEZLIN

were electrophoretically heterogeneous (data not shown). They resolved into several faint bands, which required silver staining for good visualization. Antigens, which bind to different IgG antibodies in the serum and should dissociate from them upon exposure to 1 N acetic acid during the IBP isolation, probably account for at least some of these heterogeneous protein bands. SDS-PAGE of the second main group of HPLC peaks (fractions 1-3, Fig. 3A) revealed that they contain homogeneous proteins which were moved as single bands with apparent molecular mass about 8 kDa (Fig. 3B). The first fraction of the third main group of HPLC peaks (fraction 4, Fig. 3A) was also homogeneous and moved as one major band with an apparent molecular mass of about 12 kDa, whereas the other fractions (fractions 5-7, Fig. 3A) were usually heterogeneous. N-terminal

analysis

of IgG binding proteins

Two IBP were identified by N-terminal sequencing as V,I (protein P12) and C,3 (protein P25) immunoglobulin fragments (Table 1). There were only two mismatches in the iv-terminal sequence of the protein P25 and that of human C,3 domain (residues 339-353 of the gamma chain). There was also significant homology between the protein of the peak 4 and the most common residues of the V regions of the human kappa light chains (subgroup I). The N-terminal sequences of the proteins of peaks 1 and 3 isolated by HPLC (Fig. 3A) were also determined. There was a complete homology between the protein of peak 3 and the N-terminal sequence of C3a. The N-terminal sequences of C4a and the protein of peak 1 differed by only one amino acid residue. The apparent molecular masses of the proteins of peaks 1 (- 8 kDa), 3 (- 8 kDa) and 4 (- 14 kDa) were close to those of C3a, C4a, and of immunoglobulin domains, respectively. The apparent molecular mass of protein P25 which exhibits homology to the human C,3 domain, was 25 kDa, i.e. two times that of the molecular mass of an immunoglobulin domain. This is probably due to strong noncovalent interactions between C,3 monomers, which would account for C,3 domains moving as dimers upon SDS-PAGE. Reassociation

of anaphylatoxins

with the IgG

The reassociation of anaphylatoxins with IgG was studied by dot blotting. When ‘*‘I-labeled IgG was added to C3a or C4a, immobilized on cellulose nitrate membranes, an intense binding of IgG to anaphylatoxins was observed (Fig. 4A). The binding of radioiodinated human serum albumin to anaphylatoxins (Fig. 4B) was about 4-5 times weaker than that of IgG, as determined by densitometric measurements (data not shown). The interaction of monoclonal IgG and its fragments

anti-C3a

antibodies

with

Cellulose nitrate membranes with immobilized human intact IgG, Fab and Fc were incubated with two different radioiodinated mAbs specific for C3a (D17/1 and K13/16, Fig. 5). IgG and Fab interacted well with the anti-C3a mAbs while the binding with Fc was very weak.

et al.

Proteins

separated

from human

939

IgG molecules

(a 1 (b) 2

C3a

C4a 3

4

Fig. 4. (a) Interaction of 1Z51-labeledIgG with C3a and C4a immobilized on nitrocellulose. Proteins of fraction 3 (C3a) and fraction 1 (C4a) (Fig. 3A) were immobilized on nitrocellulose membrane (10 ~1 containing 0.5 pg per point) and membranes were incubated with ‘2sI-labeled IgG (0.8 pg, 7 x 10’ cpm) in 10 ml of PBS. (b) Interaction of C3a (1 and 2) and C4a (3 and 4) immobilized on nitrocellulose with 12’I-IgG (1 and 3) and ‘251-human serum albumin (2 and 4). C3a and C4a (fractions 3 and 4, Fig. 3A) were immobilized on nitrocellulose (10 ~1 containing 2.5 pg per point) and the membranes were incubated with the ‘2SI-labeled proteins (0.8 pg, 1.8 x 10’ cpm) diluted in 10 ml PBS. Autoradiographs of dot blots are presented.

There was no interaction of the anti-C3a mAbs with commercial preparations of ovalbumin, bovine serum albumin or human serum albumin. Thus, by utilizing specific anti-C3a mAbs the presence of C3a in complexes with IgG or its fragments can be easily determined. DISCUSSION The experiments described herein demonstrated that several groups of proteins can associate with human IgG molecules. They were separated using 1 N acetic acid and 5 M GuHCl solutions. Fractionation by reverse-phase HPLC revealed three main groups of IBP. The first

W Fob

C3a Ova Fig. 5. Interaction of radioiodinated monoclonal antibodies specific for C3a with immobilized intact human IgG and its Fab and Fc fragments. IgG, Fab, Fc or ovalbumin (25 pg per point, of 5 ml 5 mg/ml solutions) and C3a (fraction 3, Fig. 3A) were immobilized on nitrocellulose and the membranes were incubated with a mixture of radioiodinated anti-C3a mAbs D17/1 and K13/16 (0.3 pg containing 1 PCi of each antibody in 1.5 ml of PBS). Autoradiographs of dot blots are presented. Ova, ovalbumin.

group of proteins was present only in acetic acid eluates at the beginning of the HPLC gradient (Fig. 2). It may contain antigens of self origin, that are present in serum and can complex with IgG antibodies. Such antigenantibody complexes would be disrupted by acetic acid treatment used during the isolation of IBP. Antibodies to self antigens were recently observed in normal mouse and human IgG preparations (Berneman et al., 1992; Saenko et al., 1992). The proteins of the second main HPLC group were identified as the C3a and C4a complement components (anaphylatoxins) (Table 1). These low molecular mass proteins are the N-terminal portions of the C3 and C4 complement components and are released upon complement activation. C3a and C4a exhibit various biological activities and are responsible for inflammatory and anaphylactoid reactions even at very low concentrations (Vogt, 1986; Hugli, 1989). Isolated anaphylatoxins can recombine with IgG molecules (Fig. 4). Potential binding sites for C3a and C4a are present on both the heavy and light chains of IgG (Nezlin and Feywald, 1992). That the major region of IgG to which C3a binds is located in the Fab fragment was demonstrated with anti-C3a mAbs (Fig. 5). Under physiological conditions the complement system is continuously activated at a low level and complement activated products, including anaphylatoxins, accumulate in serum (Opperman et al., 1992). Some of the IgG-anaphylatoxin complexes detected and identified in this study may be generated during the in vitro isolation procedure, when the concentration of anaphylatoxins increases significantly. However, it is very likely that IgG-anaphylatoxin complexes also form in vivo, since low levels of anaphylatoxins are constantly present in the circulation. Due to the ability of IgG molecules to combine with anaphylatoxins, they probably serve as

940

ROALD NEZLIN et al.

scavengers of free anaphylatoxin molecules, especially when the level of these highly active complement fragments increases in serum significantly due to compThere is probably a dynamic lement activation. equilibrium between the level of free and IgG complexed anaphylatoxin molecules in serum. Two types of immunoglobulin fragments, the V,I and C,3 domains, were identified in the third main HPLC fraction of IBP. These fragments are probably generated by the proteolytic cleavage of the corresponding immunoglobulin peptide chains. In fact human light chains can be digested by trypsin, papain, or pepsin to yield V and C halves (Karlsson et al., 1969). The kappa chains of the IgG molecule could be cleaved by proteolytic enzymes, either in vivo or during the isolation procedures. The V,I domains in these cleaved IgG molecules are associated with V, domains by noncovalent interactions, which are disrupted upon exposure to acetic acid and GuHCl. Similar events could occur with the C,3 domains. The N-terminal amino acid residue of the C,3 fragment obtained herein was alanine, which is in position 339 of the human gammachains. The amino acid residue in position 338 of these chains is lysine. Trypsin specifically catalyses the splitting of peptide bonds on the carboxylic side of lysine residues, therefore the appearance of the C,3 fragment is probably due to the action of a trypsin-like enzyme(s). Another possibility is that the V domains are synthesized by B lymphocytes at early stages of their development as separate proteins and then are combined with IgG molecules. Some of the 10” lymphocytes present in humans may synthesize C,3 domains as a result of genetic defects. However, there is no clear experimental evidence for either of the latter two hypotheses. The IBP isolated in these experiments contain also proteins that have yet to be identified. Some of them may be soluble forms of membrane proteins (Fc receptors and CD4) or serum proteins like fibronectin (Rostagno et al., 1991) and glycoprotein 60 (Sandilands et al., 1991) recently found in complexes with IgG. Gamma-globulin preparations, containing IgG, are widely used for treatment of various diseases. Further studies of IBP will elucidate whether side-effects of this therapy are caused by proteins complexed with IgG. Acknowledgements-This study was supported by the Henry Gutwirth Fund. We wish to thank Dr E. Shinar, A. Staroselsky and R. Bar-Or of the National Blood Service, Tel-Hashomer Hospital, Tel-Aviv, for kindly providing sera from patients with multiple sclerosis and B. Schick for reviewing the manuscript.

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Opperman M., Haubitz M., Quentin E. and Giitze 0. (1988) Complement activation in patients with renal failure as detected through the quantitation of fragments of the complement proteins C3, C5 and factor B. Klin. Wochenschr. 66, 857-864. Oppermann M., Hopken U. and Giitze 0. (1992) Assessment of complement activation in vivo. Immunopharmacology 24, 119-134. Oppermann M., Liebmann F. and Gijtze 0. (1987) Purification and quantitation of human C3a anaphylatoxin using monoclonal antibodies. Complement 4, 205-206. Piischel G. P., Oppermann M., Muschol W., Gotze 0. and Jungermann K. (1989) Increase of glucose and lactate output and decrease of flow by human anaphylatoxin C3a but not C5a in perfused rat liver. FEBS Letts 243, 83-87. Rostagno A. A., Frangione B. and Gold L. (1991) Biochemical studies on the interaction of fibronectin with Ig. J. Zmmun. 146, 2687-2693. Saenko V. A., Kabakov A. E. and Poverenny A. M. (1992) Hidden high-avidity anti-DNA antibodies occur in normal human gammaglobulin preparations. Immun. Letts. 34, l-6. Sandilands G. P., Ahmed A. E. E., Griffiths M. R. and Whaley K. (1990) Immunohistochemical localization of a plasma protein (glycoprotein 60) which inhibits complement-mediated prevention of immune precipitation. Immunology 70, 303-308. Vogt W. (1986) Anaphylatoxins: possible roles in disease. Complement 3, 177-188.