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Proteolytic digestion of mouse IgE
Journal of Immunological Methods, 138 (1991) 15-23
15
© 1991 ElsevierSciencePublishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100121R JIM05870
Proteolytic digestion of mouse IgE * Seiji H a b a a n d A l f r e d N i s o n o f f Rosenstiel Research Center, Department of Biology, Brandeis University, Waltham, MA 02254, U S.A.
(Received 29 November 1990, accepted 7 December1990)
Conditions are described for the preparation of F(ab')2 and Fab fragments of mouse IgE. Papain, pepsin or trypsin each produced F(ab')2 fragments with M r --- 130,000 which yielded Fab fragments on further digestion. The release of Fab fragments from F(ab')2 resulted from further cleavage of the H chain. Pepsin, and especially trypsin appear more suitable for the preparation of F(ab')2 because of the difficulty of separating a 93 kDa by-product from the F(ab')2 produced by papain. The best yields of purified Fab were obtained with papain. Rates of digestion were in the order, pepsin ----trypsin >> papain. Key words: IgE, mouse; Proteolyticdigestion; Papain; Pepsin; Trypsin
Introduction
Because of the small amounts available in serum the proteolysis of IgE has been studied exclusively by using monoclonal IgE immunoglobulins. In the case of human IgE, there have been a number of investigations of proteolysis by papain, pepsin or trypsin, and the fragments produced, including Fc, F(ab')z and Fab, have been well characterized (Bennich and Johansson, 1971). The proteolysis of rat IgE was investigated by Ellerson et al. (1978), Perez-Montfort and Metzger (1982) and Rousseaux-Prevost et al. (1983). These studies demonstrated the formation of F(ab')2 fragments after
Correspondence to: S. Haba, Rosenstiel Research Center, Brandeis UniversityWaltham, MA 02254, U.S.A. * Supported by Grants AI-22068 from the National Institutes of Health and IM607 from the American Cancer Society. Abbreviations: Ars, p-azobenzenearsonate; BGG, bovine gamma globulin; KLH, keyhole limpet hemocyanin; nlAb, monoclonal antibody; SDS-PAGE, sodium dodecylsulfatepolyacrylamidegel electrophoresis.
treatment with each of several proteolytic enzymes, including papain (Ellerson et al., 1978; Rousseaux-Prevost et al., 1983), pepsin (Ellerson et al., 1978) or trypsin (Ellerson et al., 1978; Perez-Montfort and Metzger, 1982). In addition, Rousseaux-Prevost et al. (1987) showed the presence of very small amounts of Fab fragments in a papain digest of rat IgE. The proteolysis of mouse IgE has been studied by Perez-Montfort and Metzger (1982), using trypsin as the enzyme. The major product was F(ab')2 and little if any Fab was produced. With the increasing availability of mouse IgE mAbs further information on their susceptibility to proteolytic enzymes should facilitate localization of biological activities to particular regions of the molecule. Also, we are interested in studying the fine specificity of monoclonal anti-IgE antibodies as well as the activation of IgE-specific helper T cells (Haba and Nisonoff, 1990) by defined fragments of mouse IgE. We report here on results of digestion of monoclonal mouse IgE with papain, pepsin or trypsin.
16
Materials and methods
Mice A/J, BALB/cJ and (BALB/cJx A / J ) F 1 (CAM) mice were obtained from the Jackson Laboratory, Bar Harbor, ME. Care of animals was in accordance with our institutional guidelines.
Monoclonal IgE antibodies A / J mice were inoculated i.p. with 5 ~tg KLHArs in alum (4 rag). After three or four injections at 3-4 week intervals, followed by a 5 day waiting period, mice were killed and hybridomas were prepared, using spleen cells and the non-secreting Sp2/0Ag-14 cell line (Haba and Nisonoff, 1985). Hybridomas secreting IgE anti-Ars were selected and cloned as previously described, then grown in pristane-treated CAF 1 mice. IgE mAb were affinity purified from ascites fluid on BGG-Ars-Sepharose 4B. IgE mAb used in the study were SE21.1, SE20.2, SE17.1 and 6-16E. mAb 6-16E is an IgEK switch variant prepared from the 6-16 hybridoma (IgGlx) by the method described by Muller and Rajewsky (1983).
Enzymes Crystallized papain was obtained from Worthington Biochemicals (Freehold, N J). Crystallized trypsin (type XIII: TPCK-treated) and pepsin were obtained from Sigma Co. (St. Louis, MO).
Enzymatic digestion Proteolysis with papain was carried out at 37 ° C in 0.1 M sodium phosphate buffer, pH 7.0, containing 0.01 M L-cysteine and 0.002 M EDTA. The total IgE concentration was 10-15 mg/ml and the weight ratio of papain to IgE was 1 : 100. Reactions were stopped by the addition of recrystallized iodoacetamide (final concentration, 0.02 M). Digestions with trypsin were carried out in 0.1 M Tris-HC1 buffer, pH 8.0, containing 0.01 M CaC12. The weight ratio of trypsin to protein was 1 : 50. Reactions were stopped by the addition of 4 mg of trypsin inhibitor per mg of trypsin. Digestions with pepsin were carried out in 0.1 M sodium acetate buffer, pH 4.5. The weight ratio of pepsin to protein was 1:50. Reactions were stopped by raising the pH to neutrality. Digests
were concentrated by centrifugation in a 'Centricell' tube (Polysciences, Warrington, PA), with a molecular weight cutoff of - 10,000.
Other methods SDS-PAGE was carried out with a 'Mini-Protean II' electrophoresis cell (Bio-Rad, Richmond, CA) according to the manufacturer's protocol. The concentration of polyacrylamide was 7.5% under non-reducing conditions and 12.5% under reducing conditions. Molecular weight standards were obtained from Bio-Rad (Richmond, CA). Mouse IgG1 (mAb 6-16) and the F(ab')2 and Fab fragments of mAb 6-16 were also used as standards. Gel filtration was done on Sephacryl S-200 (Pharmacia-LKB, Piscataway, N J) in borate-saline buffer, pH 8.0, ionic strength 0.16. Double diffusion in agarose gels utilized polyclonal rabbit antiIgE, prepared against monoclonal mouse IgE (TIB 142) and adsorbed with a non-specific mouse serum Ig fraction. The antibody was affinity purified on mAb SE1.3, conjugated to Sepharose 4B; the antibody was eluted with 3 M NaSCN (Haba and Nisonoff, 1985). Results
Time course of digestion of IgE with papain Fig. l a shows the effect of treatment with papain of mAb 6-16E (IgEx). The intact IgE (lane B) has a relative molecular weight (Mr) of 200,000. After 1 h treatment, the strongest band (lane C) has M r = 130,000. The M r values of the two major bands below it are 93,000 and 62,000. For reasons indicated below, we believe that the products with M r = 130,000 and 62,000 represent F(ab') 2 and Fab fragments, respectively and that the F(ab')2 is not a simple dimer of Fab. With increasing periods of digestion (2-8 h; columns D-G) the putative F(ab')2 fragment was further digested, whereas the 62 kDa (Fab) component reached its maximum concentration at 4 or 6 h. Very little intact IgE remained after 2 h. The SDS-PAGE patterns obtained after treatment with reducing agent (Fig. lb) will be considered later.
Characterization of fragments Fig. 2 shows the gel filtration pattern (on Sephacryl S-200) of the 6 h papain digest represented
17
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was approximately 80%. The two major bands in lanes D and E of Fig. 3a each were accompanied by smaller amounts of slightly faster moving material, suggesting some heterogeneity with respect to the precise point of cleavage by papain. These minor bands were retained during affinity purification (lane E), indicating that they also possess anti-Ars activity. Peak II of Fig. 2 contained the 62 kDa protein (lane G, Fig. 3a). This protein had specificity for Ars as shown by its affinity purification (lane H, yield - 75%). Most of the protein in peak II1 was of low molecular weight and migrated with the tracking dye (lane J). Lanes F and I contained protein from peaks I and II, respectively, that passed through the BGGArs-Sepharose column and thus lacked anti-Ars activity. For peak I (lane F) the amount of unbound protein was small (yield, 0.3% of total protein) and only 1/5 as much protein was loaded as in the other lanes. In lane I (unbound protein of peak II; yield - 1%) the M r of the major band was 43,000; its possible derivation will be considered later.
14 Fig. 1. SDS-PAGE showing the time course of digestion of IgE (mAb 6-16E) with papain, a: unreduced; b: reduced (0.5 M 2-mercaptoethanol). Lanes A and H contain standards for Me; lane B, untreated lgE; lanes C - G , IgE digested for periods of 1, 2, 4, 6 and 8 h, respectively. The numerals on the right are M r × 10 -3 values for the standards.
SDS-PA GE under reducing conditions Fig. lb shows patterns corresponding to the proteins in Fig. la, but obtained under reducing conditions; i.e., the same materials were loaded into corresponding lanes in Figs. la and lb. In
3.0
in Fig. 1. Four major peaks are labeled I-IV. The protein in peak IV was dialyzable and was not studied further. SDS-PAGE analyses of peaks I, II and III and of the unfractionated digest are shown in Fig. 3a. Lane B of Fig. 3a contains untreated IgE and lane C the unfractionated 6 h papain digest (same material as lane F, Fig. 1). The three major bands in lane C will be referred to as 130, 93 and 62 kDa bands. The 130 kDa and 93 kDa proteins were both eluted in peak I of Fig. 2 (lane D, Fig. 3a) which comprised approximately 30% of the total protein that was treated with papain. Both of these proteins possessed anti-Ars activity, as shown by their recovery from a BGG-Ars-Sepharose colunm (lane E of Fig. 3). The yield of the affinity purification
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Fig. 2. Gel filtration pattern obtained with a 6 h papain digest of m A b 6-16E. 200 m g of digest was concentrated by centrifugation in a 'Centricell' tube, then applied to a 600 cm 3 column of Sephacryl S-200. Each of the 150 fractions contained 3.3 ml.
18
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Fig. 3. Results of SDS-PAGE. a: unreduced; b: reduced proteins. Lanes A and K contain Mr standards. F(ab')2 and Fab standards were derived from IgG1. Lane B, undigested mAb 6-16E. Lane C, unfractionated 6 h papain digest. Lane D, peak I obtained by gel filtration of the 6 h digest (Fig. 2); E, affinity purified protein of peak I; F, protein of peak I that failed to bind to BGG-Ars-Sepharosein the affinity purification step; G, protein of peak II; H, affinity purified protein of peak II; I, protein of peak II that failed to bind to BGG-ArsSepharose during affinity purification; J, protein of peak III.
Figs. l a and lb lanes A and H contained standards of known molecular weight. About four times as much protein was loaded in lanes C - G (digested IgE) as in lane B (undigested); this applies to both Figs. l a and lb. Reduced, undigested IgE migrated as two bands with M r = 87,000 and 27,000, respectively, corresponding to H and L chains (lane B). After 1 h of digestion by papain (lane C) the reduced product showed six distinct bands (and two or three faint bands). The slowest moving major band ( - 87 kDa) corresponded to undigested H chains and the other strong band ( - 2 7 kDa) to undigested L chains. The weaker band just below the L chain (25 kDa) has not been characterized. We will consider first the two definite bands
below the H chain band (53 kDa and 41 kDa). The 53 kDa band appears to correspond to the H chain fragment present in the 130 kDa material. First, 130 kDa and 53 kDa are the strongest bands larger than L chains in the unreduced and reduced digest, respectively. Second, as the digestion proceeds (lanes D - G ) the 130 kDa and 53 kDa bands diminish proportionately in the unreduced and reduced products, respectively. Third, the gel filtration peak I (Fig. 2), which contains the 130 kDa material, yields a major band upon reduction of M r = 52,000 (lanes D or E, Fig. 3b), as well as a 27 kDa band (L chain).
Properties of the major bands in the unreduced papain digest We will consider in turn the nature of the 62 kDa, 130 kDa and 93 kDa bands in the unreduced digest (Figs. l a and 3a). The 62 kDa band evidently represents Fab fragments. It is localized in peak II after gel filtration (lane G, Fig. 3a) and has anti-Ars activity as shown by its affinity purification (lane H, Fig. 3a). It comprised approximately 16% of the total protein used in the digestion. After reduction it exhibits two chains (lanes G and H, Fig. 3b), one of which corresponds to an L chain (27 kDa); the other must be a cleavage product of the H chain (41 kDa). The sum of the M r values for H and L (69,000) agrees reasonably well with the M r of the unreduced protein (62,000; bands G or H, Fig. 3a). Despite its specificity for Ars, the 62 kDa material fails to precipitate with antigen, BGG-Ars, in a double diffusion test (Fig. 4A, well b), a property consistent with univalence. In addition, it inhibits the precipitation of F(ab')2 of IgE by BGG-Ars. This is shown by the shortening of the F(ab')2 precipitin band (well a) on the side adjacent to well b, which contains Fab. Its ability to inhibit precipitation is again consistent with univalence. During digestion (Fig. l a ) the yield of Fab is maximal after 4 - 6 h of digestion and appears to increase up to 6 h at the expense of the 130 kDa product. We propose that the 130 kDa protein is F(ab')2. This component has a somewhat larger H chain fragment than does the Fab. This is evident from lanes D and G of Fig. 3a; the two H chain digestion products have M r = 53,000 and 41,000, respectively. The 130 kDa protein has specificity
19
for Ars as shown by its affinity purification (lane E of Fig. 3a). In addition this protein (from a 6 h papain digest) forms a precipitate with BGG-Ars (Fig. 4A, well a), a property consistent with bivalence. An apparent difficulty is the poor agreement of its M r (130,000) with the sum of its two H (53,000) and two L (27,000) components; that sum is 160,000. This apparent inconsistency might be explained by variability in the effect of carbohydrate (Weber and Osborn, 1969), or of a loosening of the polypeptide structure (Springer et al., 1977) on the mobility in SDS-PAGE. In any case, the intensities of the bands in the unreduced and reduced protein (lanes D, Figs. 3a and 3b) indicate that the material that forms the 130 kDa band yields major bands of 53 kDa and 27 kDa after reduction. Also, the 27 kDa product corresponds to an L chain, as expected for a F(ab')2-1ike product. A very similar apparent discrepancy between the M r values of reduced and unreduced F(ab')2 fragments in a tryptic digest of mouse IgE was observed by Perez-Montfort and Metzger (1982).
Nature of the 93 kDa band The material in the 93 kDa band also has anti-Ars activity, as shown by its affinity purification (Fig. 3a, lane E). It is of interest that both the 130 kDa and 93 kDa bands in unreduced peak I (lane D, Fig. 3a) evidently yield 53 kDa and 27 kDa chains upon reduction. A possible explanation is that the 93 kDa product contains a corn-
plete Fab' moiety plus a portion of the second H chain. A somewhat analogous molecule is the F a b / c fragment obtained by partial digestion of mouse (Parham, 1983) or rabbit IgG (Goodman, 1965); this consists of a half-molecule plus the C terminal portion of the second H chain. In the present instance, it is proposed that one L chain and the N terminal portion of one H chain component are removed by further cleavage of F(ab')2, whereas the second H chain component (53 kDa) and L chain are not cleaved. Upon reduction, this product should yield the remaining portion of the further-cleaved 53 kDa H chain as well as the observed 53 kDa and 27 kDa chains. This is consistent with the presence of a 22 kDa band, visible below the L chain in reduced peak I (Fig. 3b, lanes D and E). Further support for this model came from an experiment (not shown) in which protein was tested from fractions on the low molecular weight (right-hand) side of peak I (Fig. 2). This material had a higher ratio of 93 to 130 kDa protein, and also gave a stronger 22 kDa band upon reduction than an equivalent amount of the protein representing all of peak I.
Absence of Fc-like material in the papain digest We did not obtain evidence for a substantial yield of Fc-like material (i.e., material of fairly high molecular weight derived from the C terminal portion of the heavy chains). This suggests that most of the C terminal regions of the H chains are
Fig. 4. Double diffusion in agarose gel. A: center well contained BGG-Ars (500/~g/ml); well d, intact mAb 6-16E (IgEr anti-Ars, 1 mg/ml); well a, affinity purified peak I (largely F(ab')2 ) of a papain digest of mAb 6-16E, 500 ~g/ml; well b, affinity purified Fab of mAb 6-16E (peak II, Fig. 2), 3 mg/ml. Wells c, e and f are empty. B: center well contained affinity purified rabbit antimouse IgE, 3 mg/ml; wells e and f, affinity purified F(ab')2-enriched fragments of mAb 6-16E, 500 # g / m l ; a and b, affinity purified Fab from mAb 6-16E, 250/~g/ml; c and d contain the 43K product that failed to bind to BGG-Ars-Sepharose (see text), 250/~g/ml.
20
cleaved by papain into small or heterogeneous fragments. However, protein of peak II (Fig. 2) contained a significant amount of 43 kDa material that failed to bind to BGG-Ars-Sepharose (lane I, Fig. 3a). This band can also be seen (faintly) in the whole digest (lane C, Fig. 3a). Upon reduction the 43 kDa protein yields a 22 kDa band (lane I), suggesting that the 43 kDa protein may be a homodimer of part of the two H chain moieties of F(ab') 2- Since the interheavy chain disulfide bonds are all in the (?.,2 domain, this dimeric material, whose molecular weight is reduced in half by reduction, is derived at least in part from this domain. Furthermore, analysis by double diffusion in agarose gel indicates that the 43 kDa 3.0
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Digestion of IgE by trypsin and pepsin
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protein is derived from F(ab')2 , since the two proteins show partial identity (Fig. 4B, wells d and e), with F(ab')2 forming a spur over the 43 kDa component. The 43 kDa component gives a line of nonidentity with Fab (Fig. 4B, wells b and c). All these data are consistent with the hypothesis that the 43 kDa protein is produced from F(ab') 2 by the further action of papain (Fig. 7). The absence of other Fc-derived material further suggests that C,4 and part or all of C,3 are degraded by papain. The diagram in Fig. 7 has interheavy chain disulfide bonds placed in accordance with amino acid sequence data (Liu, 1986). The proposed cleavage of F(ab')2 into Fab therefore requires that the interheavy chain bond closest to the N terminus be broken, presumably by the 0.01 M cysteine present during papain digestion. The double-diffusion patterns in Fig. 4B show that the F(ab')2 protein (affinity purified peak I, Fig. 2) forms a spur (line of partial identity) over Fab (affinity purified peak II) when tested against rabbit anti-IgE. This observation, the M r values for their respective H chains (53 kDa and 41 kDa), and the apparent increase of Fab at the expense of F(ab')2 as digestion proceeds (Fig. 1), all indicate that a C-terminal portion of the two H chain fragments present in F(ab')2 (principally the C,2 domains) are further cleaved from F(ab')2 by the continued action of papain. The evidence cited above indicates that the peptides released comprise the 43 kDa product.
130
140
150
Fig. 5. A: gel filtration pattern of a 30 n'fin trypsin digest (80 nag) of m A b 6-16E on Sephacryl S-200. B: gel filtration pattern of a 30 min pepsin digest of m A b 6-16E. Each fraction contained 3.2 ml. 80 m g of protein was applied to each column.
Fig. 6a shows a comparison of results of SDSPAGE on digests of IgE (mAb 6-16E) with papain (lane B), trypsin (lane C) and pepsin (lane D). The periods of digestion for the three enzymes were 6 h, 30 min and 30 min, respectively. After these time intervals no significant amounts of IgE remained undigested. The slowest-moving major band ( - 1 3 0 kDa) appears to correspond in each case to F(ab')2. Upon gel filtration the 130 kDa protein was eluted in peak I (Fig. 5A and 5B). The protein in peak I comprised approximately 45% of the total protein digested by trypsin and 41% for the peptic digest, as compared to 30% for papain. It was recovered from each digest by affinity purification on BGGArs-Sepharose (lanes E, F and G, Fig. 6a), formed
21
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Fig. 6. SDS-PAGE of proteolytic digests of mAb 6-16E; a: unreduced; b: reduced. Lanes A and N contain M r standards. F(ab')2 and Fab standards are from IgG1. Lanes B, C, and D, unfractionated digests obtained with papain (6 h), trypsin (30 min), and pepsin (30 min), respectively. E, F, and G, affinity purified protein from peak I (Figs. 2, 5A and 5B) of each of the three digests (papain, trypsin and pepsin, respectively). H, I, J, affinity purified protein from peak II of the corresponding three digests. K, protein of peak II of a papain digest (Fig. 1) that was not bound to BGG-Ars-Sepharose. L and M, proteins taken from gel-filtration fractions labeled Ia in Figs. 5A and 5B, that failed to bind to BGG-Ars-Sepharose. (The yield of such unbound material from peak II in Figs. 5-4 and 5B was too small to give visible bands.)
a precipitate with BGG-Ars in a double diffusion test (not shown) and gave bands of - 53 kDa and - 2 7 kDa upon reduction (lanes B-G, Fig. 6b). Each enzyme also produced an Fab-like fragment of M r = 62,000, 47,000 or 60,000 (for papain, trypsin or pepsin, respectively; Fig. 6a, H, I, and J). However, the yield of Fab-like material in a tryptic digest was very small (Fig. 5A, peak II). Each Fab-like fraction could be affinity purified from BGG-Ars-Sepharose (lanes H, I and J, Fig. 6a). Upon subsequent reduction each gave a 27 kDA L chain band; papain and pepsin also yielded 41 kDa and 37 kDa H chain fragments, respectively (Fig. 6b). The H chain fragment in the reduced tryptic digest shows a band of - 16 kDa,
but it is considerably weaker then the L chain band. The nature of the H chain material in the Fab-like fragment produced by trypsin is uncertain; a possibility is microheterogeneity with respect to the points of cleavage of the H chain fragment by trypsin, giving rise to a number of weak bands. Data not shown indicated that the affinity purified Fab obtained with pepsin inhibited precipitation of IgE anti-Ars by BGG-Ars (as did the Fab produced by papain; Fig. 4A, see above). The yield of Fab-like protein ( - 2% of the total) after tryptic digestion was too small to allow testing for blocking of precipitation. As already indicated, one product of papain digestion of IgE anti-Ars is a fragment of 43 kDa that lacks anti-Ars activity and is not retained by a BGG-Ars-Sepharose colunm. Lanes K, L and M of Fig. 6 (a and b) indicate that all 3 enzymes yield a similar product ( - 43 kDa) which is cleaved in half upon reduction. Lane K contained protein from gel filtration peak II of a papain digest (Fig. 2), that failed to bind to BGG-Ars-Sepharose. Lanes L and M contained similar proteins from a tryptic and peptic digest, respectively, taken from gel-filtration fractions labeled Ia (Figs. 5A and 5B). In all 3 cases F(ab')2 formed a spur and a line of partial identity with the 43 kDa protein (data shown only for the papain digest; Fig. 4B). For reasons discussed above, we believe that the 43 kDa material is largely derived from the two C,2 domains of the H chain. We should note that a small amount of precipitate formed in each peptic or tryptic digest. It redissolved on bringing the peptic digest to neutrality. In a tryptic digest the precipitate comprised approximately 2% of the absorbancy of the total protein at 280 nm (measured by dissolving the precipitate at pH 12).
Digestion of other IgE mAbs with papain The work described so far was done with mAb 6-16E. We investigated the time course of digestion by papain of three other IgEx mAb: SE17.1, SE20.2 and SE21.1. All four behaved almost identically, as indicated by SDS-PAGE, except that the digestion of F(ab')2 of SE20.2 into Fab fragments proceeded more rapidly than that of the other three IgE mAbs (not shown).
22
Discussion
Conditions for the proteolytic digestion of monoclonal mouse IgE with papain, pepsin or trypsin were investigated. The work with trypsin complements that of Perez-Montfort and Metzger (1982). Methods for the preparation of F(ab')2 or Fab fragments are described. Our results indicate that F(ab')2 can most readily be obtained in a relatively pure state from a peptic or tryptic digest. Papain is the enzyme of choice for preparing Fab. With each enzyme a major product was F(ab')2 with M r ~ 130,000. A similar result was obtained, with mouse IgE and trypsin, by Perez-Montfort and Metzger (1982). Each F(ab')2 protein could be enriched by gel filtration and affinity purified on BGG-Ars-Sepharose; each enriched product formed a precipitate in a double diffusion test with BGG-Ars, an observation consistent with bivalence. The purified F(ab')2 was contaminated with a 93,000 product, but the latter was a minor component of the products obtained after tryptic or pep tic digestion ( Fig. 6 a ). F(ab' ) 2 from a tryp tic or peptic digest could be obtained essentially free of 93K material by taking the protein from the high molecular weight edge of peak I after gelfiltration (not shown). Upon SDS-PAGE under reducing conditions the F(ab')2 product of each enzyme yielded polypeptide chains of M r - 53,000 and 27,000. The 27 kDa protein corresponds in M r to that of L chains derived from untreated IgE. (Untreated IgE yields H chains with M r = 87,000). The periods of digestion required varied for the three enzymes. Significant amounts of undigested IgE were no longer observed after digestion periods of 4-6 h, 30 min and 30 min for papain (pH 7), trypsin, (pH 8) and pepsin (pH 4.5), respectively. For IgG1 (mAb 6-16), the corresponding periods were about 2 h for papain and 8-12 h, at pH 4.1 (a lower pH), for pepsin (data not shown). Similar results were obtained with other mouse IgG1 mAbs (Lamoyi and Nisonoff, 1983). Thus, the relative susceptibilities to papain and pepsin are reversed for IgE and IgG1. None of the three enzymes tested yielded significant amounts of an Fc-like fragment. It appears that the C,4 and part or all of tl~e C,3
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Fig. 7. Suggested model for the cleavage of mouse IgE by papain. The first and second points of cleavage, yielding F(ab' ) 2 and Fab, respectively, are labeled papain (1) and (2). Note that the N terminal interheavy chain disulfide bond must be broken to release Fab fragments, in addition to the papain (2) cleavage. The 93 kDa by-product (see text) may comprise one Fab' fragment and a 22 kDa cleavage product of the second H chain.
domain is rapidly digested into small or heterogeneous products. Another product of papain digestion of IgE was Fab (62 kDa), which increased in concentration at the apparent expense of F(ab')2 as the digestion proceeded, between 2 and 6 h. The Fab could be affinity purified on BGG-Ars-Sepharose, failed to precipitate with BGG-Ars, and inhibited the precipitation of F(ab')2 with BGG-Ars, a property consistent with monovalence. During the proteolytic conversion of F(ab')2 to Fab the H chains of F(ab') 2 were further cleaved so that their M r was reduced from 53,000 to 41,000. A suggested mechanism is shown in Fig. 7. Two other important products of papain digestion have M r = 93,000 and 43,000. The 43,000 protein lacks Ars-binding capability and appears during the conversion of F(ab')2 to Fab. On reduction it yields a single 22 kDa band. In double diffusion tests the 43 kDa component gave fines of partial identity with F(ab')2 and non-identity with Fab indicating that it is cleaved from F(ab')2 during the digestion. The 43 kDa component therefore appears to be a dimer of 22 kDa peptides, derived from H chains, which is removed from F(ab')2 by the continued action of papain (Fig. 7). The 93 kDa product has not yet been well characterized. Upon reduction it yields major bands of 53 kDa (corresponding to the H chain component of F(ab')2), 27 kDa (L chain) and a small amount of 22 kDa protein. We propose that the 93 kDa product contains a complete Fab' and
23 a 22 kDa portion of the second H chain, probably derived from C,2 and C,3. It would then be somewhat related (but not identical) in structure to F a b / c of rabbit or mouse IgG which consist of one half-molecule and a part of the second H chain. F a b / c is produced by minimal digestion with papain (Goodman, 1965) or pepsin (Parham, 1983). More data are needed to prove the structure of the 93 kDa protein. Each of four IgE mAb tested behaved almost identically during papain digestion except that the digestion of F(ab')2 into Fab from one of the mAb (SE20.2) proceeded more rapidly than that of the others. Although each of the three enzymes tested yielded substantial amounts of F(ab')2, only papain gave a good yield of Fab. Fab could be detected in pepsin or trypsin digests, but only in small amounts, particularly in the tryptic digest. In addition, the M r values of the Fab fragments differed; for papain it was 62,000, pepsin 60,000, and trypsin 47,000. Each Fab fragment yielded a 27 kDa L chain; the variability in M r was due to the size of the H chain fragment, indicating that the preferential points of further cleavage of the H chain varied after the initial cleavage that yields 130 kDa F(ab')2. The Fab purified from a papain digest by gel-filtration followed by affinity purification gave a single 62 kDa band on SDS-PAGE (Fig. 3a). We can compare results obtained with mouse, human and rat IgE. In contrast with mouse IgE, the human protein yielded a prominent Fc fragment upon digestion with papain (Bennich and Johansson, 1971). In addition, only Fab and not F(ab')2 fragments were reported in the papain digest. Treatment of human IgE with pepsin or trypsin yielded F(ab')2 fragments of M r 140,000 and 103,000, respectively (as compared to 130,000 for mouse IgE), but not Fab fragments (Bennich and Johannson, 1971). Rat IgE is similar to mouse IgE in that each of the three enzymes yielded a major product (M r = 128,000-140,000), corresponding to F(ab')2 (Ellerson et al., 1978; Perez-Montfort and Metzger, 1982; Rousseaux-Provost et al., 1987). A very low
yield of a product with Mr ----60,000 was observed in a papain digest of rat igE and tentatively identified as Fab by Rousseaux-Prevost et al. (1987). References Bennich, H. and Johansson, S.G.O. (1971) Structure and function of human immunoglobulin E. Adv. Immunol. 13, 1. Ellerson, J.R., Karlsson, T. and Bennich, H. (1978) Rat IgE. Differences between free and cell-bound IgE in susceptibility to proteolytic cleavage. Protides Biol. Fluids 25, 739. Goodman, J.W. (1965) Heterogeneity of rabbit y-globulin with respect to cleavage by papain. Biochemistry 4, 2350. Haba, S. and Nisonoff, A. (1985) Quantitation of IgE antibodies by radioimmunoassay in the presence of high concentration of non-lgE antibodies of the same specificity. J. Immunol. Methods 85, 39. Haba, S. and Nisonoff, A. (1987) Production of syngeneic autoreactive monoclonal antibodies specific for isotypic determinants of IgE. J. Immunol. Methods 105, 193. Haba, S. and Nisonoff, A. (1990) Inhibition of IgE synthesis by anti-IgE: Role in long-term inhibition of IgE synthesis by neonatally administered soluble IgE. Proc. Natl. Acad. Sci. U.S.A. 87, 3363. Lamoyi, E. and Nisonoff, A. (1983) Preparation of F(ab') 2 fragments from mouse IgG of various subclasses. J. Immunol. Methods 56, 235. Liu, F.-T. (1986) Gene expression and structure of immunoglobulin epsilon chains. Crit. Rev. Immunol. 6, 47. Muller, C.E. and Rajewsky, K. (1983) Isolation of immunoglobulin class switch variants from hybridoma lines secreting anti-idiotope antibodies by sequential sublining. J. Immunol. 131, 877. Parham, P. (1983) On the fragmentation of monoclonal IgG1, IgG2a, IgG2b from BALB/c mice. J. Immunol. 131, 2895. Perez-Montfort, R. and Metzger, H. (1982) Proteolysis of soluble IgE receptor complexes: Localization of sites on IgE which interact with the Fc receptor. Mol. Immunol. 19, 1113. Rousseaux-Prevost, R., Rousseaux, J., Bazin, H. and Biserte, G. (1983) Formation of biologically inactive polymer is responsible for the thermal inactivation of rat IgE. Int. Arch. Allergy Appl. Immunol. 70, 268. Rousseaux-Prevost, R., Rousseaux, J. and Bazin, H. (1987) Studies of the IgE binding sites to rat mast cell receptor with proteolytic fragments and with a monoclonal antibody directed against epsilon heavy chain: evidence that the combining sites are located in the Cc3 domain. Mol. Immunol. 24, 187. Springer, T., Kaufman, J.F., Terhorst, C. and Strominger, J.L. (1977) Purification and structural characterization of human HLA-linked B-cell antigens. Nature 268, 213. Weber, K. and Osborn, M. (1969) The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 224, 4406.
15
© 1991 ElsevierSciencePublishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100121R JIM05870
Proteolytic digestion of mouse IgE * Seiji H a b a a n d A l f r e d N i s o n o f f Rosenstiel Research Center, Department of Biology, Brandeis University, Waltham, MA 02254, U S.A.
(Received 29 November 1990, accepted 7 December1990)
Conditions are described for the preparation of F(ab')2 and Fab fragments of mouse IgE. Papain, pepsin or trypsin each produced F(ab')2 fragments with M r --- 130,000 which yielded Fab fragments on further digestion. The release of Fab fragments from F(ab')2 resulted from further cleavage of the H chain. Pepsin, and especially trypsin appear more suitable for the preparation of F(ab')2 because of the difficulty of separating a 93 kDa by-product from the F(ab')2 produced by papain. The best yields of purified Fab were obtained with papain. Rates of digestion were in the order, pepsin ----trypsin >> papain. Key words: IgE, mouse; Proteolyticdigestion; Papain; Pepsin; Trypsin
Introduction
Because of the small amounts available in serum the proteolysis of IgE has been studied exclusively by using monoclonal IgE immunoglobulins. In the case of human IgE, there have been a number of investigations of proteolysis by papain, pepsin or trypsin, and the fragments produced, including Fc, F(ab')z and Fab, have been well characterized (Bennich and Johansson, 1971). The proteolysis of rat IgE was investigated by Ellerson et al. (1978), Perez-Montfort and Metzger (1982) and Rousseaux-Prevost et al. (1983). These studies demonstrated the formation of F(ab')2 fragments after
Correspondence to: S. Haba, Rosenstiel Research Center, Brandeis UniversityWaltham, MA 02254, U.S.A. * Supported by Grants AI-22068 from the National Institutes of Health and IM607 from the American Cancer Society. Abbreviations: Ars, p-azobenzenearsonate; BGG, bovine gamma globulin; KLH, keyhole limpet hemocyanin; nlAb, monoclonal antibody; SDS-PAGE, sodium dodecylsulfatepolyacrylamidegel electrophoresis.
treatment with each of several proteolytic enzymes, including papain (Ellerson et al., 1978; Rousseaux-Prevost et al., 1983), pepsin (Ellerson et al., 1978) or trypsin (Ellerson et al., 1978; Perez-Montfort and Metzger, 1982). In addition, Rousseaux-Prevost et al. (1987) showed the presence of very small amounts of Fab fragments in a papain digest of rat IgE. The proteolysis of mouse IgE has been studied by Perez-Montfort and Metzger (1982), using trypsin as the enzyme. The major product was F(ab')2 and little if any Fab was produced. With the increasing availability of mouse IgE mAbs further information on their susceptibility to proteolytic enzymes should facilitate localization of biological activities to particular regions of the molecule. Also, we are interested in studying the fine specificity of monoclonal anti-IgE antibodies as well as the activation of IgE-specific helper T cells (Haba and Nisonoff, 1990) by defined fragments of mouse IgE. We report here on results of digestion of monoclonal mouse IgE with papain, pepsin or trypsin.
16
Materials and methods
Mice A/J, BALB/cJ and (BALB/cJx A / J ) F 1 (CAM) mice were obtained from the Jackson Laboratory, Bar Harbor, ME. Care of animals was in accordance with our institutional guidelines.
Monoclonal IgE antibodies A / J mice were inoculated i.p. with 5 ~tg KLHArs in alum (4 rag). After three or four injections at 3-4 week intervals, followed by a 5 day waiting period, mice were killed and hybridomas were prepared, using spleen cells and the non-secreting Sp2/0Ag-14 cell line (Haba and Nisonoff, 1985). Hybridomas secreting IgE anti-Ars were selected and cloned as previously described, then grown in pristane-treated CAF 1 mice. IgE mAb were affinity purified from ascites fluid on BGG-Ars-Sepharose 4B. IgE mAb used in the study were SE21.1, SE20.2, SE17.1 and 6-16E. mAb 6-16E is an IgEK switch variant prepared from the 6-16 hybridoma (IgGlx) by the method described by Muller and Rajewsky (1983).
Enzymes Crystallized papain was obtained from Worthington Biochemicals (Freehold, N J). Crystallized trypsin (type XIII: TPCK-treated) and pepsin were obtained from Sigma Co. (St. Louis, MO).
Enzymatic digestion Proteolysis with papain was carried out at 37 ° C in 0.1 M sodium phosphate buffer, pH 7.0, containing 0.01 M L-cysteine and 0.002 M EDTA. The total IgE concentration was 10-15 mg/ml and the weight ratio of papain to IgE was 1 : 100. Reactions were stopped by the addition of recrystallized iodoacetamide (final concentration, 0.02 M). Digestions with trypsin were carried out in 0.1 M Tris-HC1 buffer, pH 8.0, containing 0.01 M CaC12. The weight ratio of trypsin to protein was 1 : 50. Reactions were stopped by the addition of 4 mg of trypsin inhibitor per mg of trypsin. Digestions with pepsin were carried out in 0.1 M sodium acetate buffer, pH 4.5. The weight ratio of pepsin to protein was 1:50. Reactions were stopped by raising the pH to neutrality. Digests
were concentrated by centrifugation in a 'Centricell' tube (Polysciences, Warrington, PA), with a molecular weight cutoff of - 10,000.
Other methods SDS-PAGE was carried out with a 'Mini-Protean II' electrophoresis cell (Bio-Rad, Richmond, CA) according to the manufacturer's protocol. The concentration of polyacrylamide was 7.5% under non-reducing conditions and 12.5% under reducing conditions. Molecular weight standards were obtained from Bio-Rad (Richmond, CA). Mouse IgG1 (mAb 6-16) and the F(ab')2 and Fab fragments of mAb 6-16 were also used as standards. Gel filtration was done on Sephacryl S-200 (Pharmacia-LKB, Piscataway, N J) in borate-saline buffer, pH 8.0, ionic strength 0.16. Double diffusion in agarose gels utilized polyclonal rabbit antiIgE, prepared against monoclonal mouse IgE (TIB 142) and adsorbed with a non-specific mouse serum Ig fraction. The antibody was affinity purified on mAb SE1.3, conjugated to Sepharose 4B; the antibody was eluted with 3 M NaSCN (Haba and Nisonoff, 1985). Results
Time course of digestion of IgE with papain Fig. l a shows the effect of treatment with papain of mAb 6-16E (IgEx). The intact IgE (lane B) has a relative molecular weight (Mr) of 200,000. After 1 h treatment, the strongest band (lane C) has M r = 130,000. The M r values of the two major bands below it are 93,000 and 62,000. For reasons indicated below, we believe that the products with M r = 130,000 and 62,000 represent F(ab') 2 and Fab fragments, respectively and that the F(ab')2 is not a simple dimer of Fab. With increasing periods of digestion (2-8 h; columns D-G) the putative F(ab')2 fragment was further digested, whereas the 62 kDa (Fab) component reached its maximum concentration at 4 or 6 h. Very little intact IgE remained after 2 h. The SDS-PAGE patterns obtained after treatment with reducing agent (Fig. lb) will be considered later.
Characterization of fragments Fig. 2 shows the gel filtration pattern (on Sephacryl S-200) of the 6 h papain digest represented
17
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21
was approximately 80%. The two major bands in lanes D and E of Fig. 3a each were accompanied by smaller amounts of slightly faster moving material, suggesting some heterogeneity with respect to the precise point of cleavage by papain. These minor bands were retained during affinity purification (lane E), indicating that they also possess anti-Ars activity. Peak II of Fig. 2 contained the 62 kDa protein (lane G, Fig. 3a). This protein had specificity for Ars as shown by its affinity purification (lane H, yield - 75%). Most of the protein in peak II1 was of low molecular weight and migrated with the tracking dye (lane J). Lanes F and I contained protein from peaks I and II, respectively, that passed through the BGGArs-Sepharose column and thus lacked anti-Ars activity. For peak I (lane F) the amount of unbound protein was small (yield, 0.3% of total protein) and only 1/5 as much protein was loaded as in the other lanes. In lane I (unbound protein of peak II; yield - 1%) the M r of the major band was 43,000; its possible derivation will be considered later.
14 Fig. 1. SDS-PAGE showing the time course of digestion of IgE (mAb 6-16E) with papain, a: unreduced; b: reduced (0.5 M 2-mercaptoethanol). Lanes A and H contain standards for Me; lane B, untreated lgE; lanes C - G , IgE digested for periods of 1, 2, 4, 6 and 8 h, respectively. The numerals on the right are M r × 10 -3 values for the standards.
SDS-PA GE under reducing conditions Fig. lb shows patterns corresponding to the proteins in Fig. la, but obtained under reducing conditions; i.e., the same materials were loaded into corresponding lanes in Figs. la and lb. In
3.0
in Fig. 1. Four major peaks are labeled I-IV. The protein in peak IV was dialyzable and was not studied further. SDS-PAGE analyses of peaks I, II and III and of the unfractionated digest are shown in Fig. 3a. Lane B of Fig. 3a contains untreated IgE and lane C the unfractionated 6 h papain digest (same material as lane F, Fig. 1). The three major bands in lane C will be referred to as 130, 93 and 62 kDa bands. The 130 kDa and 93 kDa proteins were both eluted in peak I of Fig. 2 (lane D, Fig. 3a) which comprised approximately 30% of the total protein that was treated with papain. Both of these proteins possessed anti-Ars activity, as shown by their recovery from a BGG-Ars-Sepharose colunm (lane E of Fig. 3). The yield of the affinity purification
I
1
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90 I 0 0 I10 120 FRACTION NUMBER
130
140
i50
Fig. 2. Gel filtration pattern obtained with a 6 h papain digest of m A b 6-16E. 200 m g of digest was concentrated by centrifugation in a 'Centricell' tube, then applied to a 600 cm 3 column of Sephacryl S-200. Each of the 150 fractions contained 3.3 ml.
18
(o)
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Fig. 3. Results of SDS-PAGE. a: unreduced; b: reduced proteins. Lanes A and K contain Mr standards. F(ab')2 and Fab standards were derived from IgG1. Lane B, undigested mAb 6-16E. Lane C, unfractionated 6 h papain digest. Lane D, peak I obtained by gel filtration of the 6 h digest (Fig. 2); E, affinity purified protein of peak I; F, protein of peak I that failed to bind to BGG-Ars-Sepharosein the affinity purification step; G, protein of peak II; H, affinity purified protein of peak II; I, protein of peak II that failed to bind to BGG-ArsSepharose during affinity purification; J, protein of peak III.
Figs. l a and lb lanes A and H contained standards of known molecular weight. About four times as much protein was loaded in lanes C - G (digested IgE) as in lane B (undigested); this applies to both Figs. l a and lb. Reduced, undigested IgE migrated as two bands with M r = 87,000 and 27,000, respectively, corresponding to H and L chains (lane B). After 1 h of digestion by papain (lane C) the reduced product showed six distinct bands (and two or three faint bands). The slowest moving major band ( - 87 kDa) corresponded to undigested H chains and the other strong band ( - 2 7 kDa) to undigested L chains. The weaker band just below the L chain (25 kDa) has not been characterized. We will consider first the two definite bands
below the H chain band (53 kDa and 41 kDa). The 53 kDa band appears to correspond to the H chain fragment present in the 130 kDa material. First, 130 kDa and 53 kDa are the strongest bands larger than L chains in the unreduced and reduced digest, respectively. Second, as the digestion proceeds (lanes D - G ) the 130 kDa and 53 kDa bands diminish proportionately in the unreduced and reduced products, respectively. Third, the gel filtration peak I (Fig. 2), which contains the 130 kDa material, yields a major band upon reduction of M r = 52,000 (lanes D or E, Fig. 3b), as well as a 27 kDa band (L chain).
Properties of the major bands in the unreduced papain digest We will consider in turn the nature of the 62 kDa, 130 kDa and 93 kDa bands in the unreduced digest (Figs. l a and 3a). The 62 kDa band evidently represents Fab fragments. It is localized in peak II after gel filtration (lane G, Fig. 3a) and has anti-Ars activity as shown by its affinity purification (lane H, Fig. 3a). It comprised approximately 16% of the total protein used in the digestion. After reduction it exhibits two chains (lanes G and H, Fig. 3b), one of which corresponds to an L chain (27 kDa); the other must be a cleavage product of the H chain (41 kDa). The sum of the M r values for H and L (69,000) agrees reasonably well with the M r of the unreduced protein (62,000; bands G or H, Fig. 3a). Despite its specificity for Ars, the 62 kDa material fails to precipitate with antigen, BGG-Ars, in a double diffusion test (Fig. 4A, well b), a property consistent with univalence. In addition, it inhibits the precipitation of F(ab')2 of IgE by BGG-Ars. This is shown by the shortening of the F(ab')2 precipitin band (well a) on the side adjacent to well b, which contains Fab. Its ability to inhibit precipitation is again consistent with univalence. During digestion (Fig. l a ) the yield of Fab is maximal after 4 - 6 h of digestion and appears to increase up to 6 h at the expense of the 130 kDa product. We propose that the 130 kDa protein is F(ab')2. This component has a somewhat larger H chain fragment than does the Fab. This is evident from lanes D and G of Fig. 3a; the two H chain digestion products have M r = 53,000 and 41,000, respectively. The 130 kDa protein has specificity
19
for Ars as shown by its affinity purification (lane E of Fig. 3a). In addition this protein (from a 6 h papain digest) forms a precipitate with BGG-Ars (Fig. 4A, well a), a property consistent with bivalence. An apparent difficulty is the poor agreement of its M r (130,000) with the sum of its two H (53,000) and two L (27,000) components; that sum is 160,000. This apparent inconsistency might be explained by variability in the effect of carbohydrate (Weber and Osborn, 1969), or of a loosening of the polypeptide structure (Springer et al., 1977) on the mobility in SDS-PAGE. In any case, the intensities of the bands in the unreduced and reduced protein (lanes D, Figs. 3a and 3b) indicate that the material that forms the 130 kDa band yields major bands of 53 kDa and 27 kDa after reduction. Also, the 27 kDa product corresponds to an L chain, as expected for a F(ab')2-1ike product. A very similar apparent discrepancy between the M r values of reduced and unreduced F(ab')2 fragments in a tryptic digest of mouse IgE was observed by Perez-Montfort and Metzger (1982).
Nature of the 93 kDa band The material in the 93 kDa band also has anti-Ars activity, as shown by its affinity purification (Fig. 3a, lane E). It is of interest that both the 130 kDa and 93 kDa bands in unreduced peak I (lane D, Fig. 3a) evidently yield 53 kDa and 27 kDa chains upon reduction. A possible explanation is that the 93 kDa product contains a corn-
plete Fab' moiety plus a portion of the second H chain. A somewhat analogous molecule is the F a b / c fragment obtained by partial digestion of mouse (Parham, 1983) or rabbit IgG (Goodman, 1965); this consists of a half-molecule plus the C terminal portion of the second H chain. In the present instance, it is proposed that one L chain and the N terminal portion of one H chain component are removed by further cleavage of F(ab')2, whereas the second H chain component (53 kDa) and L chain are not cleaved. Upon reduction, this product should yield the remaining portion of the further-cleaved 53 kDa H chain as well as the observed 53 kDa and 27 kDa chains. This is consistent with the presence of a 22 kDa band, visible below the L chain in reduced peak I (Fig. 3b, lanes D and E). Further support for this model came from an experiment (not shown) in which protein was tested from fractions on the low molecular weight (right-hand) side of peak I (Fig. 2). This material had a higher ratio of 93 to 130 kDa protein, and also gave a stronger 22 kDa band upon reduction than an equivalent amount of the protein representing all of peak I.
Absence of Fc-like material in the papain digest We did not obtain evidence for a substantial yield of Fc-like material (i.e., material of fairly high molecular weight derived from the C terminal portion of the heavy chains). This suggests that most of the C terminal regions of the H chains are
Fig. 4. Double diffusion in agarose gel. A: center well contained BGG-Ars (500/~g/ml); well d, intact mAb 6-16E (IgEr anti-Ars, 1 mg/ml); well a, affinity purified peak I (largely F(ab')2 ) of a papain digest of mAb 6-16E, 500 ~g/ml; well b, affinity purified Fab of mAb 6-16E (peak II, Fig. 2), 3 mg/ml. Wells c, e and f are empty. B: center well contained affinity purified rabbit antimouse IgE, 3 mg/ml; wells e and f, affinity purified F(ab')2-enriched fragments of mAb 6-16E, 500 # g / m l ; a and b, affinity purified Fab from mAb 6-16E, 250/~g/ml; c and d contain the 43K product that failed to bind to BGG-Ars-Sepharose (see text), 250/~g/ml.
20
cleaved by papain into small or heterogeneous fragments. However, protein of peak II (Fig. 2) contained a significant amount of 43 kDa material that failed to bind to BGG-Ars-Sepharose (lane I, Fig. 3a). This band can also be seen (faintly) in the whole digest (lane C, Fig. 3a). Upon reduction the 43 kDa protein yields a 22 kDa band (lane I), suggesting that the 43 kDa protein may be a homodimer of part of the two H chain moieties of F(ab') 2- Since the interheavy chain disulfide bonds are all in the (?.,2 domain, this dimeric material, whose molecular weight is reduced in half by reduction, is derived at least in part from this domain. Furthermore, analysis by double diffusion in agarose gel indicates that the 43 kDa 3.0
I
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90 I00 I10 120 FRACTION NUMBER
41
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Digestion of IgE by trypsin and pepsin
I
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90 I 0 0 I10 120 FRACTION NUMBER
protein is derived from F(ab')2 , since the two proteins show partial identity (Fig. 4B, wells d and e), with F(ab')2 forming a spur over the 43 kDa component. The 43 kDa component gives a line of nonidentity with Fab (Fig. 4B, wells b and c). All these data are consistent with the hypothesis that the 43 kDa protein is produced from F(ab') 2 by the further action of papain (Fig. 7). The absence of other Fc-derived material further suggests that C,4 and part or all of C,3 are degraded by papain. The diagram in Fig. 7 has interheavy chain disulfide bonds placed in accordance with amino acid sequence data (Liu, 1986). The proposed cleavage of F(ab')2 into Fab therefore requires that the interheavy chain bond closest to the N terminus be broken, presumably by the 0.01 M cysteine present during papain digestion. The double-diffusion patterns in Fig. 4B show that the F(ab')2 protein (affinity purified peak I, Fig. 2) forms a spur (line of partial identity) over Fab (affinity purified peak II) when tested against rabbit anti-IgE. This observation, the M r values for their respective H chains (53 kDa and 41 kDa), and the apparent increase of Fab at the expense of F(ab')2 as digestion proceeds (Fig. 1), all indicate that a C-terminal portion of the two H chain fragments present in F(ab')2 (principally the C,2 domains) are further cleaved from F(ab')2 by the continued action of papain. The evidence cited above indicates that the peptides released comprise the 43 kDa product.
130
140
150
Fig. 5. A: gel filtration pattern of a 30 n'fin trypsin digest (80 nag) of m A b 6-16E on Sephacryl S-200. B: gel filtration pattern of a 30 min pepsin digest of m A b 6-16E. Each fraction contained 3.2 ml. 80 m g of protein was applied to each column.
Fig. 6a shows a comparison of results of SDSPAGE on digests of IgE (mAb 6-16E) with papain (lane B), trypsin (lane C) and pepsin (lane D). The periods of digestion for the three enzymes were 6 h, 30 min and 30 min, respectively. After these time intervals no significant amounts of IgE remained undigested. The slowest-moving major band ( - 1 3 0 kDa) appears to correspond in each case to F(ab')2. Upon gel filtration the 130 kDa protein was eluted in peak I (Fig. 5A and 5B). The protein in peak I comprised approximately 45% of the total protein digested by trypsin and 41% for the peptic digest, as compared to 30% for papain. It was recovered from each digest by affinity purification on BGGArs-Sepharose (lanes E, F and G, Fig. 6a), formed
21
(a)
ABCDEFG
H IJ
KLMN
T~Gt -" F(ab')2 -
92.5 66 45
Fab-
(b)
A BC D E F G H I I
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E--II
-
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~-31
21 iii~ii!'~!iiii
Fig. 6. SDS-PAGE of proteolytic digests of mAb 6-16E; a: unreduced; b: reduced. Lanes A and N contain M r standards. F(ab')2 and Fab standards are from IgG1. Lanes B, C, and D, unfractionated digests obtained with papain (6 h), trypsin (30 min), and pepsin (30 min), respectively. E, F, and G, affinity purified protein from peak I (Figs. 2, 5A and 5B) of each of the three digests (papain, trypsin and pepsin, respectively). H, I, J, affinity purified protein from peak II of the corresponding three digests. K, protein of peak II of a papain digest (Fig. 1) that was not bound to BGG-Ars-Sepharose. L and M, proteins taken from gel-filtration fractions labeled Ia in Figs. 5A and 5B, that failed to bind to BGG-Ars-Sepharose. (The yield of such unbound material from peak II in Figs. 5-4 and 5B was too small to give visible bands.)
a precipitate with BGG-Ars in a double diffusion test (not shown) and gave bands of - 53 kDa and - 2 7 kDa upon reduction (lanes B-G, Fig. 6b). Each enzyme also produced an Fab-like fragment of M r = 62,000, 47,000 or 60,000 (for papain, trypsin or pepsin, respectively; Fig. 6a, H, I, and J). However, the yield of Fab-like material in a tryptic digest was very small (Fig. 5A, peak II). Each Fab-like fraction could be affinity purified from BGG-Ars-Sepharose (lanes H, I and J, Fig. 6a). Upon subsequent reduction each gave a 27 kDA L chain band; papain and pepsin also yielded 41 kDa and 37 kDa H chain fragments, respectively (Fig. 6b). The H chain fragment in the reduced tryptic digest shows a band of - 16 kDa,
but it is considerably weaker then the L chain band. The nature of the H chain material in the Fab-like fragment produced by trypsin is uncertain; a possibility is microheterogeneity with respect to the points of cleavage of the H chain fragment by trypsin, giving rise to a number of weak bands. Data not shown indicated that the affinity purified Fab obtained with pepsin inhibited precipitation of IgE anti-Ars by BGG-Ars (as did the Fab produced by papain; Fig. 4A, see above). The yield of Fab-like protein ( - 2% of the total) after tryptic digestion was too small to allow testing for blocking of precipitation. As already indicated, one product of papain digestion of IgE anti-Ars is a fragment of 43 kDa that lacks anti-Ars activity and is not retained by a BGG-Ars-Sepharose colunm. Lanes K, L and M of Fig. 6 (a and b) indicate that all 3 enzymes yield a similar product ( - 43 kDa) which is cleaved in half upon reduction. Lane K contained protein from gel filtration peak II of a papain digest (Fig. 2), that failed to bind to BGG-Ars-Sepharose. Lanes L and M contained similar proteins from a tryptic and peptic digest, respectively, taken from gel-filtration fractions labeled Ia (Figs. 5A and 5B). In all 3 cases F(ab')2 formed a spur and a line of partial identity with the 43 kDa protein (data shown only for the papain digest; Fig. 4B). For reasons discussed above, we believe that the 43 kDa material is largely derived from the two C,2 domains of the H chain. We should note that a small amount of precipitate formed in each peptic or tryptic digest. It redissolved on bringing the peptic digest to neutrality. In a tryptic digest the precipitate comprised approximately 2% of the absorbancy of the total protein at 280 nm (measured by dissolving the precipitate at pH 12).
Digestion of other IgE mAbs with papain The work described so far was done with mAb 6-16E. We investigated the time course of digestion by papain of three other IgEx mAb: SE17.1, SE20.2 and SE21.1. All four behaved almost identically, as indicated by SDS-PAGE, except that the digestion of F(ab')2 of SE20.2 into Fab fragments proceeded more rapidly than that of the other three IgE mAbs (not shown).
22
Discussion
Conditions for the proteolytic digestion of monoclonal mouse IgE with papain, pepsin or trypsin were investigated. The work with trypsin complements that of Perez-Montfort and Metzger (1982). Methods for the preparation of F(ab')2 or Fab fragments are described. Our results indicate that F(ab')2 can most readily be obtained in a relatively pure state from a peptic or tryptic digest. Papain is the enzyme of choice for preparing Fab. With each enzyme a major product was F(ab')2 with M r ~ 130,000. A similar result was obtained, with mouse IgE and trypsin, by Perez-Montfort and Metzger (1982). Each F(ab')2 protein could be enriched by gel filtration and affinity purified on BGG-Ars-Sepharose; each enriched product formed a precipitate in a double diffusion test with BGG-Ars, an observation consistent with bivalence. The purified F(ab')2 was contaminated with a 93,000 product, but the latter was a minor component of the products obtained after tryptic or pep tic digestion ( Fig. 6 a ). F(ab' ) 2 from a tryp tic or peptic digest could be obtained essentially free of 93K material by taking the protein from the high molecular weight edge of peak I after gelfiltration (not shown). Upon SDS-PAGE under reducing conditions the F(ab')2 product of each enzyme yielded polypeptide chains of M r - 53,000 and 27,000. The 27 kDa protein corresponds in M r to that of L chains derived from untreated IgE. (Untreated IgE yields H chains with M r = 87,000). The periods of digestion required varied for the three enzymes. Significant amounts of undigested IgE were no longer observed after digestion periods of 4-6 h, 30 min and 30 min for papain (pH 7), trypsin, (pH 8) and pepsin (pH 4.5), respectively. For IgG1 (mAb 6-16), the corresponding periods were about 2 h for papain and 8-12 h, at pH 4.1 (a lower pH), for pepsin (data not shown). Similar results were obtained with other mouse IgG1 mAbs (Lamoyi and Nisonoff, 1983). Thus, the relative susceptibilities to papain and pepsin are reversed for IgE and IgG1. None of the three enzymes tested yielded significant amounts of an Fc-like fragment. It appears that the C,4 and part or all of tl~e C,3
L
VL
I
H
VH
I ''~SCtl
H
I~
eL~
PAPAIN (2) PAPAIN {I)
[
1
I, C~2 ,I S S S IW¢ I I S S S iI i iI
C,=5
I
C,4
I
PAPAIN (2l PAPAIN (I)
Fig. 7. Suggested model for the cleavage of mouse IgE by papain. The first and second points of cleavage, yielding F(ab' ) 2 and Fab, respectively, are labeled papain (1) and (2). Note that the N terminal interheavy chain disulfide bond must be broken to release Fab fragments, in addition to the papain (2) cleavage. The 93 kDa by-product (see text) may comprise one Fab' fragment and a 22 kDa cleavage product of the second H chain.
domain is rapidly digested into small or heterogeneous products. Another product of papain digestion of IgE was Fab (62 kDa), which increased in concentration at the apparent expense of F(ab')2 as the digestion proceeded, between 2 and 6 h. The Fab could be affinity purified on BGG-Ars-Sepharose, failed to precipitate with BGG-Ars, and inhibited the precipitation of F(ab')2 with BGG-Ars, a property consistent with monovalence. During the proteolytic conversion of F(ab')2 to Fab the H chains of F(ab') 2 were further cleaved so that their M r was reduced from 53,000 to 41,000. A suggested mechanism is shown in Fig. 7. Two other important products of papain digestion have M r = 93,000 and 43,000. The 43,000 protein lacks Ars-binding capability and appears during the conversion of F(ab')2 to Fab. On reduction it yields a single 22 kDa band. In double diffusion tests the 43 kDa component gave fines of partial identity with F(ab')2 and non-identity with Fab indicating that it is cleaved from F(ab')2 during the digestion. The 43 kDa component therefore appears to be a dimer of 22 kDa peptides, derived from H chains, which is removed from F(ab')2 by the continued action of papain (Fig. 7). The 93 kDa product has not yet been well characterized. Upon reduction it yields major bands of 53 kDa (corresponding to the H chain component of F(ab')2), 27 kDa (L chain) and a small amount of 22 kDa protein. We propose that the 93 kDa product contains a complete Fab' and
23 a 22 kDa portion of the second H chain, probably derived from C,2 and C,3. It would then be somewhat related (but not identical) in structure to F a b / c of rabbit or mouse IgG which consist of one half-molecule and a part of the second H chain. F a b / c is produced by minimal digestion with papain (Goodman, 1965) or pepsin (Parham, 1983). More data are needed to prove the structure of the 93 kDa protein. Each of four IgE mAb tested behaved almost identically during papain digestion except that the digestion of F(ab')2 into Fab from one of the mAb (SE20.2) proceeded more rapidly than that of the others. Although each of the three enzymes tested yielded substantial amounts of F(ab')2, only papain gave a good yield of Fab. Fab could be detected in pepsin or trypsin digests, but only in small amounts, particularly in the tryptic digest. In addition, the M r values of the Fab fragments differed; for papain it was 62,000, pepsin 60,000, and trypsin 47,000. Each Fab fragment yielded a 27 kDa L chain; the variability in M r was due to the size of the H chain fragment, indicating that the preferential points of further cleavage of the H chain varied after the initial cleavage that yields 130 kDa F(ab')2. The Fab purified from a papain digest by gel-filtration followed by affinity purification gave a single 62 kDa band on SDS-PAGE (Fig. 3a). We can compare results obtained with mouse, human and rat IgE. In contrast with mouse IgE, the human protein yielded a prominent Fc fragment upon digestion with papain (Bennich and Johansson, 1971). In addition, only Fab and not F(ab')2 fragments were reported in the papain digest. Treatment of human IgE with pepsin or trypsin yielded F(ab')2 fragments of M r 140,000 and 103,000, respectively (as compared to 130,000 for mouse IgE), but not Fab fragments (Bennich and Johannson, 1971). Rat IgE is similar to mouse IgE in that each of the three enzymes yielded a major product (M r = 128,000-140,000), corresponding to F(ab')2 (Ellerson et al., 1978; Perez-Montfort and Metzger, 1982; Rousseaux-Provost et al., 1987). A very low
yield of a product with Mr ----60,000 was observed in a papain digest of rat igE and tentatively identified as Fab by Rousseaux-Prevost et al. (1987). References Bennich, H. and Johansson, S.G.O. (1971) Structure and function of human immunoglobulin E. Adv. Immunol. 13, 1. Ellerson, J.R., Karlsson, T. and Bennich, H. (1978) Rat IgE. Differences between free and cell-bound IgE in susceptibility to proteolytic cleavage. Protides Biol. Fluids 25, 739. Goodman, J.W. (1965) Heterogeneity of rabbit y-globulin with respect to cleavage by papain. Biochemistry 4, 2350. Haba, S. and Nisonoff, A. (1985) Quantitation of IgE antibodies by radioimmunoassay in the presence of high concentration of non-lgE antibodies of the same specificity. J. Immunol. Methods 85, 39. Haba, S. and Nisonoff, A. (1987) Production of syngeneic autoreactive monoclonal antibodies specific for isotypic determinants of IgE. J. Immunol. Methods 105, 193. Haba, S. and Nisonoff, A. (1990) Inhibition of IgE synthesis by anti-IgE: Role in long-term inhibition of IgE synthesis by neonatally administered soluble IgE. Proc. Natl. Acad. Sci. U.S.A. 87, 3363. Lamoyi, E. and Nisonoff, A. (1983) Preparation of F(ab') 2 fragments from mouse IgG of various subclasses. J. Immunol. Methods 56, 235. Liu, F.-T. (1986) Gene expression and structure of immunoglobulin epsilon chains. Crit. Rev. Immunol. 6, 47. Muller, C.E. and Rajewsky, K. (1983) Isolation of immunoglobulin class switch variants from hybridoma lines secreting anti-idiotope antibodies by sequential sublining. J. Immunol. 131, 877. Parham, P. (1983) On the fragmentation of monoclonal IgG1, IgG2a, IgG2b from BALB/c mice. J. Immunol. 131, 2895. Perez-Montfort, R. and Metzger, H. (1982) Proteolysis of soluble IgE receptor complexes: Localization of sites on IgE which interact with the Fc receptor. Mol. Immunol. 19, 1113. Rousseaux-Prevost, R., Rousseaux, J., Bazin, H. and Biserte, G. (1983) Formation of biologically inactive polymer is responsible for the thermal inactivation of rat IgE. Int. Arch. Allergy Appl. Immunol. 70, 268. Rousseaux-Prevost, R., Rousseaux, J. and Bazin, H. (1987) Studies of the IgE binding sites to rat mast cell receptor with proteolytic fragments and with a monoclonal antibody directed against epsilon heavy chain: evidence that the combining sites are located in the Cc3 domain. Mol. Immunol. 24, 187. Springer, T., Kaufman, J.F., Terhorst, C. and Strominger, J.L. (1977) Purification and structural characterization of human HLA-linked B-cell antigens. Nature 268, 213. Weber, K. and Osborn, M. (1969) The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 224, 4406.