Different susceptibility of subclasses of rat IgG2 to tryptic digestion

Immunochemistry 1973,Vol. 10 pp. 651-652. PergamonPress. PrintedinGreat Britain

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D I F F E R E N T S U S C E P T I B I L I T Y OF S U B C L A S S E S OF RAT IgG2 TO TRYPTIC DIGESTION R. S. N E Z L I N , M. Yu. K R I L O V and O, V. R O K H L I N Institute of Molecular Biology. Academy of Sciences of the USSR, Moscow 117312, USSR (Received 7 February 1973)

Two subclasses of rat IgG2 with different properties of the Fc fragments were recently described (Bloch et al., 1968; Nussenzweig and Binaghi, 1965). The main subclass lgG2a was isolated by chromatography on DEAEcellulose equilibrated with 0.005 M phosphate buffer, pH 8.0. By this method, however, it is not possible to separate the second subclass lgG2b since it is eluted together with lgG2a after a small decrease of pH and an increase of molarity of the buffer (for example, by 0-01 M phosphate buffer, pH 7,5) (Stechschulte and Austen, 1970). It is well known that the subclasses of human lgG possess different susceptibility to proteolysis, in particular to trypsin digestion (Gergely et al., 1972). We have employed the difference in resistance to trypsin digestion for the differentiation of IgG2 subclasses of rat. It was observed that IgG2a was not digested by trypsin while lgG2b was split into 3.5 S fragments. Immunoglobulins G2 were isolated from sera of three inbred rat strains (MSU, August, WAG), which have different allotypic variants of light chains (Rokhlin et al., 1971; Vengerova et al., 1972). Serum proteins were precipitated with sodium sulphate at 18% saturation, were dialysed against several changes of 0-005 M phosphate buffer, pH 8-0 and were applied to a DEAEcellulose column (DE32, Whatman), equilibrated with the same buffer. The protein eluted with the starting buffer appeared as two unresolved peaks (fraction 1). After subsequent application of 0.01 M phosphate buffer, pH 7.5 more protein was eluted as a single peak (fraction II). Fractions were concentrated by Diaflo ultrafiltration (Amicon). Both fractions were analysed by immunoelectrophoresis and double diffusion in agar gel with rabbit antisera against a total fraction of rat lgG, which had been isolated on DEAE cellulose equilibrated with 0.0175 M phosphate buffer, pH 6.3 (Fig. 1 and 2). In immunoelectrophoresis fraction ! usually produced one major arc and sometimes small second arc, both of which possessed lgG2 mobility. Fraction I1 in all cases, regularly produced two precipitin arcs also with IgG2 mobility (see Fig. I, a). As revealed by double diffusion in agar gel all bands of fraction I fused in a reaction of identity with bands of fraction I1 (Fig. 2a). In some experiments fraction 11 produced only one broad band. Fraction 1 produced one major band and one or two minor ones. There were no significant differences by either technique between the preparations isolated from the various rat strains. The isolated fractions were digested by trypsin (Calbiochem, DCC-treated) in a 1 : 100 ratio to protein for 16 hr at 37°C in 0.05 M Tris-HCI buffer, pH 8.0. After addition of an equimolar amount of trypsin inhibitor (Soybean, Serva) the hydrolysates were fractionated on a 1.5 × 90

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Fig. 1. Immunoelectrophorteic analysis of fraction II (a), non-digested protein and the 3.5 fragments isolated by gelfiltration from the tryptic digest of fraction II (b and c). cm column (Pharmacia) of Sephadex G-200 using a 0.05 M Tris-HC1, pH 8.0 in 0.28 M NaCI. The sedimentation analysis ~as performed using a Spinco Model E analytical ultracentrifuge. According to results of sedimentation analysis (Fig. 3) the majority of fraction 1 was not digested by trypsin. In the same conditions a considerable portion of fraction 11 was split into 3.5 S fragments. These data were confirmed by the geltiltration experiments (Fig. 4). The main portion of the protein of fraction i was eluted in the position of non-digested lgG and only a very small amount of protein appeared later (Fig. 4a). Different results were obtained after gelfiltration of the digested fraction il. In this case about one third of the protein was eluted in the position similar to that of ovalbumin (Fig. 4b) and hence represented 3.5 S fragments. Both non-digested protein and 3.5 S fragments were separately pooled, concentrated by Diaflo ultrafiltration and investigated by immunoelectrophoresis and double diffusion in agar gel (Figs. 1 and 2). After digestion, the major precipitation bands of fractions I and 11 were retained but the minor band(s) were absent or seen as very faint lines (Fig. lb), The major bands of both fractions gave spurs with the precipitation band of the 3.5 S tryptic fragments (Fig. 2b). The band of the fragments fused in a reaction of identity with minor band(s) of fraction I. The band of fraction 11 fused

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(b) Fig. 2. Double diffusion analysis. Centre well: rabbit antiserum against total fraction of rat lgG (see test): (a) 1-fraction I; 2-non-digested protein, isolated by gelfiltration from the tryptic digest of fraction 1; 3-nondigested protein, isolated by gelfiltration from the tryptic digest of fraction II and 4-fraction II. (b) I-fraction II; 2-tryptic 3.5 S fragments isolated from the digest of fraction II; 3-fraction I and 4-non-digested protein isolated from the digest of fraction II.

Fig. 4. Gel filtration patterns of fractions l(top) and 11 (bottom) digested by trypsin on a Sephadex G-200 column (1.5 x 9 0 c m , Pharmacia). Elution with 0.05 M Tris-HCI, pH 8.0 in 0.28 M NaCI, Fractions 3 ml. Ov and lgG-positions of ovalbumin and IgG. as subclass lgG2a. The other one which was present only in small amounts in fraction 1 and in considerably larger amounts in fraction II is split by trypsin into 3.5 S fragments (subclass lgG2b). The differences in susceptibility to trypsin may be accounted for by the peculiar primary structure of the hinge regions of both gammachain subclasses. By means of chromatography on DEAE-cellulose it is not possible to separate IgG2a and lgG2b. But by the combination of the trypsin digestion and subsequent gelfiltration of the digest one can isolate IgG2a and tryptic 3.5 S fragments of lgG2b. Tryptic digestion may also be used as a simple method for typing of lgG2 myeloma proteins. It is necessary however to mention that more than two subclasses of rat lgG2 may exist. This is suggested by the finding of three precipitation bands in fraction I in some experiments. A c k n o w l e d g e m e n t - T h i s work was partly supported by World Health Organization.

Fig. 3. Ultracentrifugal analysis of fractions 1 (bottom) and II (top) of rat IgG2 after trypsin digestion. Analysis was performed at 20°C in 0.1M NaCI and 0 . 1 M TrisHC1 buffer, pH 8.0. The pattern was recorded after 40 rain at 56000 rev/min. The direction of sedimentation is from left to right. in a reaction of partial identity with the non-digested protein of the same fraction (Fig. 2). The data obtained indicate the existence of two main variants of rat lgG2. One of them is resistant to trypsin action and quantitatively predominates. According to the tentative classification (BIoch et al., 1968) it is nominated

REFERENCES

Bloch K. J., Morse H. C. and Austen K. F. (1968) J. lmmun. 101,650. Gergely J., Medgyesi G. A., Wang A. G. and Fudenberg H. H, (1972) Immunochemistry 9, 589. Nussenzweig V. and Binaghi R. A. (1965) Int. Archs A Ilergy 27, 355. Rokhlin O. V., Vengerova T. 1. and Nezlin R. S. 11971) Immunochemistry 8, 525. Stecbschulte D. J. and Austen K. F. (1970) J. Immun. 104, 1052. Vengerova T. 1., Rokhlin O. V. and Nezlin R. S. (1972) lmmunochemistry 9, 1239,