lmm~ochemistry, 1975, Vol. 12, pp. 839-842. Pergamon Press. Printed in Great Britain

COMMUNICATIONS TO THE EDITORS I M M U N O L O G I C A L A N D CHEMICAL STUDIES O N SUB-FRACTIONS OF CARCINOEMBRYONIC ANTIGEN G. T. R O G E R S , F. S E A R L E a n d M. WASS Department of Medical Oncology, Charing Cross Hospital, London W.6 8RF, England (First received 24 February 1975; in revised form 8 April 1975) Abstract--The results of this study have shown that carcinoembryonic antigen (CEA) and also its desialylated product can be separated by con A affinity chromatography into three immunologicaily related sub-fractions. Each fraction reacted with established anti-CEA antisera (anti-A) and also with antisera specific for a determinant, common to both CEA and a normal glycoprotein NGP. Carbohydrate analysis and circular dichroism studies have revealed chemical differences between the fractions. Removal of sialic acid from CEA appears to have no effect on its binding to either antibody or concanavalin A and the presence of the latter, up to a 40-fold excess, does not inhibit the binding of CEA to antibody. It is now firmly established (von Kleist, 1973; Darcy et al., 1973) that carcinoembryonic antigen (CEA) possesses at least two different immunogenic groups; a unique group only in CEA-like molecules (group A) and a second determinant (group C) common to CEA and the glycoprotein NGP which is found in normal and tumoral tissues (Mach and Pasztaszeri, 1972). Double diffusion studies (Darcy et al., 1973; Tomita et al., 1974) have revealed an antiserum which reacts only with CEA (anti-A) and another which reacts equally well with CEA and N G P (anti-C). These antisera and also the cross-reactive antiserum (aA-aC) are frequently obtained by inoculating animals with highly purified CEA preparations from which N G P has been separated (G. T. Rogers, unpublished result). Evidence has recently been reported (Darcy et al., 1973; Rogers et al., 1974; Newman et al., 1974) that CEA may possess additional antigenic determinants indicating further immunological heterogeneity. In previous studies on the heterogeneity of CEA (Rogers et al., 1974) we have used concanavalin A affinity chromatography to discriminate differences in the carbohydrate moiety. Three antigenically related sub-fractions were obtained. Fraction 1 was unbound and fractions 2 and 3 were eluted with 2% and 10% methyl glucoside respectively. These studies have been confirmed in part by Yamamoto et al. (1974) who showed that preparations of CEA obtained by the method of Krupey (1972) from liver metastases of rectal, gastric and pancreatic tumours, could be fractionated by a similar technique into unbound and bound fractions. Here we present further immunological and chemical studies on these components. MATERIALS AND METHODS

Isolation of CEA CEA was extracted from liver metastases of colonic tumour tissue with perchioric acid as described by Coligan et al. (1972) and the extract purified by Sepharose 4B and Sephadex G-200 chromatography.

buffer was applied and fraction 2 collected. Fraction 3 was eluted using 10% methyl glucoside. Each fraction was dialysed against several changes of distilled water and concentrated by ultra-filtration using an Amicon PM 10 membrane. The recovery of protein in each fraction was 10, 50 and 20% respectively. Preparation of antisera Antisera 225 and 226 were prepared in rabbits by giving multiple site intradermal injections of CEA fraction 2 distributed in the flank. Each rabbit received 200 #g of protein in 0"5 ml of 0"9% saline and emulsified with an equal volume of Freund's complete adjuvant. A similar booster was given approximately 40 days later and trial bleeds commenced after a further i0 days. Antisera were absorbed with freeze-dried extracts of normal colon (40 mg/ml of antiserum), normal liver (40 mg/ml), normal spleen and normal human plasma (20 mg/ml). Antiserum 204 was obtained by inoculating rabbits with purified unfractionated CEA using a similar procedure and after absorbing it was shown to produce a reaction of identity with CEA and NGP (anti-C). 'Ace' 10 and G P are established anti-CEA antisera donated by Drs. C. W. Todd and J. T. Tomita respectively. Carbohydrate analysis of CEA sub-fractions Neutral sugars including fucose were determined by the orcinol-sulphuric acid method by measurement at 505 nm and galactose was estimated by the method of Wallenfels and Kurz (1962) after hydrolysis in 2 N HC1 at 100° for 4 hr. Fucose was estimated by the method of Disehe and Shettles (1948) and hexosamine determined by the method of Elson and Morgan (1933) after hydrolysis in 2 N HC1 at 90 ° for 3 hr. Further hydrolysis, up to 9 hr did not release significant further amounts of hexosamine. Sialic acid was determined by the method of Ayala et al. (1951).

Con A affinity chromatography Treatment of CEA with neuraminidase A solution containing CEA (10-50 rag) was dialysed A solution of CEA (25 mg in 5 rnl of water) was dialyzed against 0.1 M sodium acetate buffer (pH 6) containing against 0'2 M sodium citrate buffer (pH 5.5) containing I M NaC1, 1 0 - 3 M CaC12, 1 0 - a M MgCl2 and 1 0 - a M 10 mM CaCl 2 and then treated with Vibrio cholerae neurMnC12. The solution was then applied to a column of con aminidase in the ratio of 1 unit per 10 #g of CEA. The A Sepharose (Pharmacia), bed volume 60 ml, and eluted reaction was allowed to proceed for approximately 24 hr with the above buffer. After collecting the unbound frac- at 37°C followed by dialysis against distilled water. The tion 1 a 2% solution of methyl glucoside in the above solution was concentrated to 5 ml. 839

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(b)

(c)

(d)

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(e)

Fig. 1. Double diffusion reactions between CEA fractions 1, 2 and 3 obtained by con A affinity chromatography and various antisera. T-anti-CEA antiserum (anti-A) donated by Dr. J. T. Tomita. Antisera 225 and 226 were prepared as described in the text. Absorbed antiserum 174-anti-CEA antiserum known from previous studies (Rogers et al., 1974) to react with CEA (A-group) and an additional antigen (B-group) found in CEA fraction 2. NGP purified preparation separated from perchloric acid extracts of colonic tumours during the purification of CEA. The centre wells of (d) and (e) contain absorbed antisera 226 and 204 respectively (see text), the latter antiserum has previously been prepared using purified CEA and shown to contain antibodies to a determinant common to CEA and NGP. All antigens were tested at a concentration of approximately 1.0 mg/ml.

Double diffusion studies

Double diffusion reactions were carried out in 1"5% agar containing 0'9% NaC1. The wells, 4 mm diameter, were placed 10 mm apart. Circular dichroism spectra o f C E A fractions These were measured in 0.9% saline on a Cary 61 recording spectropolarimeter.

RESULTS Antisera 226 produced a single precipitin line on double diffusion against fraction 2 (1 mg/ml) identical to a line given by established monospecific anti-A antisera ('ace' 10 and GP), Fig. la. Early bleeds of antiserum 225 however produced an additional reaction with fraction 2. This reaction was shown to be identical to that given by the Bgroup antigen previously shown to be present in fraction 2 (Rogers et al., 1974), Fig. lb. After absorption of antiserum 225 with a CEA fraction devoid of detectable Bgroup activity (e.g. fraction 1 from con A affinity chromatography) a weak anti-B antiserum was obtained which reacted only with CEA fraction 2. It appears that the Bgroup is a much weaker immunogen in rabbits than the A-group. On testing the three con A fractions and purified NGP by double diffusion with various anti-CEA antisera we have been able to rationalise our previous results in terms of the CEA determinants described above; Anti-A antisera (226, 'ace' 10 and GP) produced a line of identity with all three fractions but not with NGP (Figs. lc and ld). An antiserum of the type which reacts both with CEA and NGP produced a line of identity with all fractions

and with NGP (Fig. le) showing that the C-group determinant is present in each of the fractions. Anti-NGP antiserum, absorbed with purifed CEA and shown to react only with the specific determinant on NGP (X-group) only reacted with fraction 3 provided this was obtained from a semi-purified CEA preparation (prior to G-200 chromatography). Fraction 3 obtained from highly purified CEA on the other hand did not react with absorbed anti-NGP, Fig. 2a. These results show that the A-group determinant is present in at least three fractions of CEA and that NGP if present in the preparation is isolated in fraction 3. Analysis has revealed differences in the carbohydrate composition between the con A fractions obtained from purified CEA (see Table 1). The most striking differences are the large carbohydrate/protein ratio and the higher content of sialic acid and hexosamine in the unbound fraction compared to the bound fractions. In agreement with previous studies on CEA (Darcy et al., 1973), fractions 1 and 2 both show double-humped arcs extending from the beta to the alpha region in immunoelectrophoresis using anti-A antiserum, Fig. 2b. Since extensive heterogeneity of the CEA polypeptide chain is unlikely (Terry et al., 1972; 1974) this finding implied that these fractions contain subspecies of CEA containing varying amounts of sialic acid. Fraction 3 containing the least amount of sialic acid per mg protein, shows the slowest anionic mobility. In comparison the arc for NGP however is weakly cationic (lowest well, Fig. 2b). Extensive isoelectric heterogeneity of CEA (Turberville et al., 1973; Coligan et al., 1973) was also found to be largely due to variability in sialic acid content. We have now demonstrated that desialylated CEA can also be fractionated by con A affinity chromatography into three antigenically active fractions reacting with anti-A

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significant change in the protein recovered in each fraction suggesting that coneanavalin A binding sites are unlikely to be exposed by desialylation of the unbound fraction. The extent, if any, to which additional binding sites are exposed in the desialylated bound fractions 2 and 3 is not known although the same concentrations of methyl glucoside (2 and 10~o respectively) are required for their elution. The above results are consistent with the fact that sialic acid in CEA is attached to penultimate galactose (Westwood et al., 1974) which is known not to bind to concanavalin A (Sharon and Lis, 1969). In order to ascertain that the con A binding sites of fraction 2 are not implicated in the antigen-antibody binding, thus confirming the results of Chu et al., (1974) for unfractionated CEA, a pseudo-standard curve was set up in which concanavalin A competed with anti-CEA antiserum ('ace' 36) for 112~ labelled fraction 2. No inhibition of the binding of label in the presence of concanavalin A, over a range of concentrations up to 40-fold excess, was found. Further studies in order to determine the relative affinities of the con A fractions for anti-A antibodies and the use of these fractions in radioimmunoassay are in progress and will be reported separately. DISCUSSION

(b) Fig. 2(a). Reaction of antiserum 226 (anti-A) with fraction 3 prepared from highly purified CEA from which NGP had been removed. (b) Immunoelectrophoresis (pH 8'5 barbitone buffer, 0.05 M) of CEA fractions obtained from semi-purified CEA against antiserum 226 and anti-NGP. The arc due to reaction of the specific determinant in N G P (X-group) in fraction 3 is partly obscured. (c) Reaction of antiserum 226 with fractions 1, 2 and 3 obtained by con A affinity chromatography of desialylated purified CEA. antiserum 226 (Fig. 2c), and also with an antiserum which reacts both with CEA and NGP, anti-C, directly demonstrating that residual heterogeneity exists in the carbohydrate moiety. Sialic acid determinations on the dialysed fractions showed that all measurable sialic acid had been released by neuraminidase treatment. Ultra-violet absorption measurements at 280 nm on the fractions, before and after neuraminidase treatment indicated that there is no Table I. Carbohydrate estimations on sub-fractions of purified CEA expressed as mg per mg protein CEA fractions 1

Neutral sugar Galactose Fucose Hexosamine Sialic acid Carbohydrate a 0 'b

0c

1.18 0.42 0.37 0.76 0.17 70 -89-4 - 9744

2

0.39 0' 11 0.11 0"17 0"07 42 -63.1 - 6878

3

0.51 0"19 0.11 0'14 0"03 44 -31-0 - 3379

"Protein estimated by Lowry's method. Circular dichroism data, expressed as ellipticity/molecular weight 0' were based on concentrations of Lowry protain. ¢0 values were calculated assuming a mean residue weight of 109.

Our results so far show that purified CEA can be fractionated into at least three glycoproteins differing in their carbohydrate moiety. Although the content of sialic acid varies between the fractions, its removal does not destroy the antigenic determinants (A- and C-groups) nor apparently alter the binding properties of the CEA fractions to immobilized concanavalin A. In view of the stereochemical specificity of concanavalin A (Sharon and Lis, 1969) and the probable absence of glucose in CEA (Terry et al., 1974) it would appear that free hydroxyl groups at the C-3, C-4 and C-6 positions of terminal non-reducing :t-mannose or ct-N-acetylglucosamine or internal ~t-l-2 linked mannose are sufficiently exposed only in fractions 2 and 3 to allow significant binding. In the unbound fraction, which contains the largest amount of carbohydrate per molecule of CEA, it is likely that con A binding groups are either modified at C-3, C-4 or C-6 by linkage to non-binding sugars such as fucose or galactose, beta-linked at the anomeric carbon or that the binding is considerably restricted by steric hindrance. In a study with Dreiding models it is shown that the binding site, particularly if this is internal 1-2 linked mannose, requires considerable freedom from neighbouring residues so that it can approach the immobilized concanavalin A molecule. Circular dichroism o f C E A fractions

Further differences between the unbound and bound fractions are reflected in the circular dichroism spectra although interpretation is difficult because of the combined effects of the peptide bonds (Ross and Jirgenson, 1968) and the acetamido chromophore in N-acetylglueosamine (Stone, 1969). Bands due to transitions in the aromatic spectral region are not observed but the two bound fractions show a negative dichroic band at 212-214 nm (see Table 1 and Fig. 3). These bands are unlikely to be due to a high content of or-helix which would give a long-wavelength n-n* Cotton effect at 222 nm, but may be correlated with a more random protein conformation or content of E-structure (Ross and Jirgenson, 1968; Doi and Jirgenson, 1970). Consistent with this interpretation is the finding (Coligan et al., 1973) that CEA contains high levels of acidic and hydroxy amino acids and proline which destabilise an alpha helical structure. In the unbound fraction the dichroic maximum is at 206 nm with a shoulder at 214 nm. The larger ellipticity (see Table 1) may be partly due to the high content of N-acetylglucosamine, the acetamido

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Fig. 3. Circular dichroism spectra of CEA fractions obtained from purified CEA. group of which is probably held in a rigid conformation by substitution at the C-3 or C-4 positions of the sugar or sterically restricted by the larger number of sugar residues per molecule of CEA in this fraction.

Acknowledoements--We thank Professor W. Klyne and Dr. P. M. Scopes of Westfield College London for their kind cooperation and facilities for circular dichroism and Dr. J. T. Tomita for the G P anti-CEA antiserum. The authors also gratefully acknowledge the skilful technical help of Mrs. J. Wood and Mr. P. McClean. This work was aided by the Medical Research Council. REFERENCES

Ayala W., Moore L. V. and Hess E. L. (1951) 3. clin. Invest. 30, 781.

Chu T. M., Holyoke E. D. and Murphy G. P. (1974) Cancer Res. 34, 212. Coligan J. E., Henkart P. A, Todd C. W. and Terry W. D. (1973) lmmunochemistry 10, 591. Coligan J. E., Lautenschleger J. T., Egan M. L. and Todd C. W. (1972) Immunochemistry 9, 377. Darcy D. A., Turberville C. and James R. (1973) Br. J. Cancer 28, 147. Dische Z. and Shettles L. R. (1948) J. biol. Chem. 175, 595. Doi E. and Jirgensons B. (1970) Biochemistry 9, 1066. Elson L. A. and Morgan W. T. J. (1933) Biochem. d. 27, 1824. yon Kleist S. (1973) Annls Inst. Pasteur, Paris 124, 589. Krupey J., Wilson T., Freedman S. O. and Gold P. (1972) Immunochemistry 9, 617. Mach J. P. and Pusztaszeri G. (1972) lmmunochemistry 9, 1031. Newman E. S., Petras S. E., Georgiadis A. and Hansen H. J. (1974) Cancer Res. 34, 2125. Rogers G. T., Searle F. and Bagshawe K. D. (1974) Nature, Lond. 251, 519. Ross D. L. and Jirgensons B. (1968) J. biol Chem. 243, 2829. Sharon N. and Lis H. (1969) Science 177, 749. Stone A. L. (1969) Biopolymers 7, 173. Terry W. D., Henkart P. A., Coligan J. E. and Todd C. W. (1972) J. exp. Med. 136, 200. Terry W. D., Henkart P. A., Coligan J. E. and Todd C. W. (1974) Transplantation (Rev.) 20, 100. Tomita, J. T., Safford J. W. and Hirata A. A. (1974) Immunology 26, 291. Turberville C., Darcy D. A., Laurence D. J. R., Johns E. W. and Neville A. M. (1973) Immunochemistry 10, 841. Wallenfels K. and Kurz G. (1962) Biochem Z. 335, 559. Westwood J. H., Bessell E. M., Bukhari A. M., Thomas P. and Walker J. M. (1974) Immunochemistry 11, 811. Yamamoto T., Gotoda S. and Kosaki G. (1974) Int. Cancer Congress Abst. XI, 1, 360.

Immunological and chemical studies on sub-fractions of carcinoembryonic antigen.

lmm~ochemistry, 1975, Vol. 12, pp. 839-842. Pergamon Press. Printed in Great Britain COMMUNICATIONS TO THE EDITORS I M M U N O L O G I C A L A N D CH...
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