Immunochemical studies on mouse myeloma proteins with specificity for dextran or for levan

ImmunochemistTy, 1972, Vol. 9, pp. 535-544. Pergamon Press. Printed in Great Britain

I M M U N O C H E M I C A L STUDIES ON MOUSE MYELOMA P R O T E I N S W I T H S P E C I F I C I T Y FOR DEXTRAN OR FOR LEVAN* ARNE LUNDBLAD?, RICHARD STELLER, ELVIN A. KABAT, JUDITH W. HIRST, MARTIN G. WEIGERT~: and MELVIN COHN Departments of Microbiology, Neurology and Human Genetics and Development, Columbia University, The Neurological Institute, Presbyterian Hospital, New York and the Salk Institute for Biological Studies, San Diego, California, U.S.A. (Received 24 September 1971) A b s t r a c t - T w o mouse myeloma proteins have been studied immnnochemically. One has

specificity for c~(1 --, 3) linked dextrans and inhibition studies with c~(1 -o 3) glucosyloligosaccharides (nigerodextrins) indicate that the size of the combining site is that of a pentasaccharide. The other protein reacts with levan and to a lesser extent with inulin. The best inhibitor was a terminal non-reducing chain of two/3 (2 -o 1) linked fructofuranosyl residues plus an additional/3 linkage either to carbon 6 of glucose or to carbon 1 of fructose. INTRODUCTION Monoclonal myeloma proteins and macroglobulins with antibody activity are of great value in studying the nature of the antibody combining site. Recently both human and mouse myeloma proteins with specificity for various antigens such as dinitrophenyl substituted proteins (Ashman and Metzger, 1969; Eisen et al., 1967, 1968; Jaffe et al., 1969, 1971; Terry etal., 1970a, b; Warner and Ovary, 1970), y globulins (Metzger, 1967; Stone and Metzger, 1969), serum lipoproteins (Beaumont, 1967; Beaumont and Lorenzelli, 1967) phosphoryl choline (Potter and Lieberman, 1970), purine and pyrimidine substituted proteins (Schubert et al., 1968) nucleic acids (Schubert et al., 1970), streptolysin (Zettervall, 1967), pneumococcal C polysaccharide (Cohn, 1967; Potter, 1968), lipopolysaccharides (Potter, 1970), dextran (Leon et al., 1970), levan (Grey et al., 1971) red blood cell antigens (Harboe, 1965) and non-reducing terminal residues of N-acetyl-D-glucosamine (Vicari et al., 1970) have been reported. A large number of myeloma proteins derived from plasma cell tumors induced in the inbred BALB/c strain of mouse have been screened for their *Aided by grants from the National Science Foundation (GB-8341 and GB-25686) to Dr. E. A. Kabat and from the National Institutes of Health (A-105875 and CA-05213) to Dr. Melvin Cohn and a General Research Support grant of the United States Public Health Service to Columbia University. ?International postdoctoral research fellow (1969-1971) of the U.S. Public Health Service and Fellow of the American Scandinavian Foundation. Present address: Institute of Medical Chemistry, University of Uppsala, Uppsala, Sweden. :~American Cancer Society Faculty Research Award No. PRA-59. 535

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A. LUNDBLAD et al.

ability to react with antigens. Protein J558, an IgA myeloma protein which precipitates best with dextrans linked a (1 --* 3) has now been studied immunochemically and has also been compared with ot (1 --* 3) specific human antidextran. Another myeloma protein J606, recently shown to belong to the new IgG3 class (Grey et al., 1971) has been shown to react with levan and, to a lesser extent, with inulin. A terminal non-reducing chain of two/3 (2 ~ 1) fructofuranosyl residues plus an additional 13linkage gave the best inhibition. MATERIALS AND METHODS Isolation 0fJ558 and J606 myeloma proteins and purification of J606. The J558 and J606 proteins were purified and characterized as previously described (Grey et al., 1971; Weigert et al., 1970). Antisera. Lat2 was from an individual immunized with dextran B 1355-S-4, (57 per cent 1 --~ 6; 34 per cent 1 --* 3-like and 9 per cent 1 ~ 4-like linkages). The antiserum was absorbed with dextran N 236 (96 per cent 1,6 and 4 per cent 1,3 like linkages) to remove antibodies of a (1 --~ 6) specificity. 36/xg dextran N 236 were added per ml of Lat2 serum. The absorbed Lat2 antiserum precipitated with dextran B1355-S-4 and not with dextran N 236. Antigens. The dextrans and levans used in this study were previously described (Allen and Kabat, 1957; Goodman and Kabat 1964; Kabat, 1961). The proportion of 1 --* 6; 1 --* 3-like and 1 --~ 4-like linkages in the different dextrans used are listed in Fig. 1. Inulin was from Nutritional Biochemicals. Mono and oligosaccharides. Various mono and oligosaccharides were described earlier (Goodman and Kabat, 1964; Mage and Kabat, 1963). Since sucrose is known to contain dextran (Hehre and Sugg, 1942; Neill et al., 1939, 1941), both sucrose and fructose were dialyzed and recrystaUized from alcohol before use. lr-fructosylsucrose (/3Dfructofuranosyl (2 ~ 1)/3vfructofuranosyl (2 ~ 1 ) aDglucopyranoside) was a gift from Dr. J. S. D. Bacon, Department of Biochemistry, Aberdeen, Scotland; and neokestose, /3Dfructofuranosyl (2 ~ 6) aoglucopyranosyl(1 ~ 2)/3Dfructofuranoside, from Dr. D. Gross, Tate and Lyle Research Laboratory, Ravensbourne, Kent, England. The di-, tri- and tetrasaccharides,/3Dfructofuranosyl (2 ~ 6) Dglucopyranose (1F1G),/3Dfructofuranosyl (2 ~ 1) /3Dfructofuranosyl (2 ~ 6) Dglucopyranose (2F1G) and /3Dfructofuranosyl(2 ~ 1)/3ofructofuranosyl(2 ~ 1) /3Dfructofuranosyl (2 --* 6) Dglucopyranose (3F1G) were obtained from Dr. F. Arcamone, Instituto Ricerche Farmitalia, Milano, Italy (Arcamone et al., 1970). Their corresponding alditols were obtained after reduction with sodium borohydride. The reaction was stopped by adding Dowex 50 H + in excess. Borate was removed by addition of methanol and evaporation; this was carried out five times. The ot (1 ~ 3) linked series of glucose oligosaccharides (nigerodextrins) was provided by Dr. I. R. Johnston, University College, London, England (Johnston, 1965). The tetramer(~oglucopyranosyl (1 --> 3) aDglucopyranosyl (1 "-> 4) otDglucopyranosyl (1 --> 3) otDglucopyranose) was a gift from Dr. J. H. Nordin, University of Massachusetts, Amherst, Mass. (Tung and Nordin, 1970). The branched oligosaccharides 33-aoglucosylisomaltotetraose, 43-aDglucosylisomaltotetraose, 34-aDglucosylisomalto -

537

Mouse Myeloma Proteins

hexaose and 44-aDglucosylisomaltohexaose* have previously been described (Bourne et al., 1963; Torii et al., 1966). Immunochemical methods Quantitative precipitin and inhibition studies were done by a microprecipitin technique (Kabat, 1961) using about 1--8/.tg of antibody N per determination. Total N in the washed precipitates was measured by the ninhydrin method (Schiffman et al., 1964). Fluorescence quenching was performed according to the method of Eisen (Eisen, 1964; Eisen and Siskind, 1964; Velick et al., 1960) using an AmincoBowman spectrophotofluorometer equipped with a cell jacket thermostated at 25°C. A square quartz semimicro cuvette having a 5 mm light path was used. 5/zl aliquot portions of sugar solution were added sequentially to 250/~1 of the purified J606 myeloma protein at a concentration of 19.5 tzg N/ml and the fluorescence measured. Correction for dilution was made by reading fluorescence in a blank titration and subtracting. The solubility of dextran B1355S-4-J558 and Hestrin levan B-J606 precipitates was studied using the same amount of antigen and antiserum at equivalence in total volumes of 0.25, 0.5, 0.75 and 1"0 ml. The solubility of each immune precipitate was calculated (Kabat, 1961). The values obtained for the dextran-J558 system were 1.1, 0.87 and 0.85/zg N/ml saline respectively and for the levan-J606 system 1-4, 0.93 and 1.0 respectively. The values are in the same range previously found for carbohydrate-human antibody reactions (Feizi and Kabat, 1971; Kabat, 1961). The effect of temperature was also investigated. Dextran B1355S-4 and levan P6 were each set up in quadruplicate with J558 and J606 protein respectively near the point of maximum precipitation and the precipitates were kept at 4°C for 5 days, washed and treated as follows: (1) Analyzed for N, (2) suspended in 0"25 ml of saline at 0°C for 1 hr, centrifuged at 0°C and analyzed, (3) suspended in 0.25 ml of saline at 37°C for 1 hr, centrifuged at 37°C and the precipitate analyzed, (4) suspended in 0.25 ml of saline at 37°C for 1 hr, cooled to 0°C for 1 hr, centrifuged at 0°C and analyzed. In the dextran B-1355 S-4-J558 system no significant differences in the nitrogen values were obtained. In the levan P6-J606 system, however, tube 3 gave a value about 40 per cent lower than

*The structures of these oligosaccharides may be represented as follows: G, Vglucopyranosyl; Gr, reducing vglucose; ---~,a (1 --* 6) link; t , a (1 ~ 3) link; ~, a (1 --* 4) link 3~c~nglucosylisomaltotetraose

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G G~G~G~G~G~Gr

538

A. LUNDBLAD et al.

tubes 1, 2 and 4, indicating that this precipitate is m u c h more soluble at 37°C than at 0°C (Feizi and Kabat, 1971). RESULTS T h e ability of different dextrans to precipitate J558 myeloma protein is given in Fig. 1 together with their proportions of 1 ~ 6; 1 ~ 3-like and 1 ~ 4-like linkages. Dextrans with a high proportion of a (1 --* 3)-like linkages (B 1355-S-4, B 1498-S and B 1501-S) were best while those with a low content of a (1 ~ 3)-like linkages and a high content of a (1 ~ 6) and/or ot (1 ~ 4)-like linkages precipitated poorly. B 742 C-3R with a relatively high proportion of both ot (1 ~ 3)like and ot (1 ~ 4)-like linkages was intermediate.

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Fig. 1. Quantitative precipitin curves of mouse myeloma serum J558 with various dextrans. Figure 2 gives results of inhibition assays. T h e best inhibitors f o u n d were the ot (1---* 3) linked series of glucose oligosaccharides (nigerodextrins). Nigeropentaose and a mixture of nigerohexa- and heptaose gave maximal inhibition of about 65 per cent and 50 per cent inhibition was reached with about 0.4 micromoles. T h e two tetraoses aDGlc(1 ~ 3)~DGlc(I ~ 3)aDGIc(I ~ 3)DGlc and aDGlc(I ~ 3)otDGlc(l ~ 4)otDGlc(l ~ 3)oGlc were indistinguishable and slightly less active; maximal inhibition reached about 60 per cent with 50 per cent inhibition at about 0"6 micromole. Nigerotriose also gave a m a x i m u m of 60 per cent inhibition but for 50 per cent inhibition about 0"7 micromole was required. Nigerose was considerably less active reaching only 40 per cent inhibition. All other mono- and oligosaccharides tested were even less active; 40 per cent inhibition requiring 80 micromoles of glucose or methyl aDglucoside.

539

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Fig. 2. Inhibition by various oligosaccharides of precipitation of mouse myeloma protein J558 by dextran. Maltose and the two branched oligosaccharides, 3 4- and 44-aDglucosylisomaltohexaose gave about 20 per cent inhibition. Inhibition with the different nigerodextrins was also studied in a dextranhuman antidextran system using an antiserum which by absorption was made more specific by removal of the a (1 ---> 6) antidextran. The results are given in Fig. 3. The nigerodextrins were found to be much less potent as inhibitors in this system as compared to the myeloma protein. Nigerose was the best inhibitor but reached only 30 per cent inhibition. None of the higher nigerodextrins was significantly better than nigerose. Considerably better as inhibitor was methyl aDglucoside reaching about 55 per cent inhibition and the four branched oligosaccharides that were tested. The most active was 34-aDglucosylisomaltohexaose which gave 90 per cent inhibition; 0.4 micromoles gave 50 per cent inhibition. The other three branched oligosaccharides tested ranged between 65 and 80 per cent maximal inhibition, and were more potent per micromole than methyl ctDglucoside. The precipitation of serum J606 with various levans and with inulin is seen in Fig. 4. All levans except perennial rye grass levan precipitated approximately the same amount of nitrogen. Hestrin levans A and B, and native levan as well as levan P6 behaved very similarly and reached equivalence with about 8/Lg antigen. With levan B 523 Fr M, levan B 512 PP2 Fr B and levan 512 Fr E, 3, 5 and 9/~g antigen was required, respectively, to precipitate 50 per cent of the maximum nitrogen. Perennial rye grass levan which, in contrast to the other levans, has a low molecular weight (about 5000) gave negligible precipitation. Inulin, also with molecular weight around 5000, precipitated much less than did the levans, and less than that reported previously for J606 (Grey et al., 1971).

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Inhibition studies with different mona- and oligosaccharides of the levan P6-J606 reaction in serum and with purified J606 protein are seen in Fig. 5. The two oligosaccharides 2F1G and 3F1G were the most potent inhibitors and were

Mouse Myeloma Proteins

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Fig. 5. Inhibition by various mono- and oligosaccharides of prec~ltatlon" " ~ of mouse myeloma protein J606 by levan. indistinguishable. They reached 100 per cent inhibition with about 19-20 nanomoles, and 50 per cent inhibition with about 6-7 nanomoles. After reduction to the corresponding alditols, reduced 3F1G retained its activity while the reduced 2F1G was about 5 times less active on a molar basis. The other mono- and oligosaccharides tested were considerably less active. They all showed inhibition in the range of 103 to 105 nanomoles. Of these, the best inhibitors were neokestose and turanose followed by melezitose, 1F1G, 1F-fructosylsucrose, raffinose, sucrose, fructose and glucose. Fructose, 2F1G and 3F1G were studied as inhibitors using both whole serum and purified J606 protein using approximately the same amount of specific precipitable nitrogen and at equivalence. No significant differences were seen. The purified J606 protein was studied in a spectrophotofluorometer. Its fluorescence excitation and emission spectra were similar to those of most proteins with an excitation maximum at 285 nm and an emission maximum at 345 nm. Five sequential additions of 3F1G (10/~g/5/.~1) and sucrose (355/~g/5/~1) to the protein did not cause any quenching of the fluorescence when corrected for dilution. DISCUSSION The J558 myeloma protein has a precipitin reaction pattern with different dextrans similar to that obtained with human antidextran with o~(1--> 3) specificity (Allen and Kabat, 1959). This new myeloma protein also shows some similarity to the recently described MOPC 104 E serum (Leon et al., 1970) which also possessed ot (1 --* 3) specificity. The best inhibitors found for the human a(1---> 3) specific antidextran (Torii et al., 1966) were the branched ol (1 ---* 3) and ol (1 --> 4) glucosyl oligo-

542

A. LUNDBLAD et al.

saccharides of the isomaltose series (Bourne et al., 1963). T h e y were better than nigerose which was the only c o m p o u n d of the nigerodextrin series then available. We are now able to confirm these results with the a (1 --* 3) specific Lat2 serum and f u r t h e r m o r e , by including the different nigerodextrins, it is demonstrated that all of these are poor inhibitors, none being significantly better than nigerose. T h e findings suggest that the specificity involves a glucosyl branch on an isomaltosyl chain. W h e t h e r this branch is attached a (1 --~ 3) or a (1 ~ 4) does not seem to have m u c h influence. T h e inhibition results with the new J558 protein are in sharp contrast to the h u m a n system since the two branched oligosaccharides tested are very poor inhibitors. T h e results, however, resemble those obtained with MOPC 104 E (Leon et al., 1970). With both, the nigerodextrins were the best inhibitors but with MOPC 104 E no significant difference in inhibiting capacity was seen between nigerotriose, -tetraose and -pentaose. In the dextran B 1498 S-J558 system the nigeropentaose and hexa-heptaose mixture are definitely better than the two tetraoses tested indicating that the size of the J558 site might be about that of a pentasaccharide while in MOPC 104 E it is probably no larger than a trisaccharide. T h e order of inhibition of the dextran-MOPC 104 E reaction by alinked glucosyloligosaccharides is 1 ~ 3 ~> 1 ~ 6 > 1 ~ 2 > 1 ~ 4 while with J558 the corresponding values would be 1 ~ 3 ~ > 1 ~ 4 ~> 1--~2 or 1 ~ 6 since both isomaltose and kojibiose were completely inactive in amounts up to about 10-15/zmoles and maltose showed slight activity (20 per cent) using between 1-2/~moles. T h e J606 protein has recently been described (Grey et al., 1971) and shown to belong to a new subclass in the mouse-IgG 3. T h e protein was f o u n d to exhibit binding activity for levan, a property which was localized to the Fab fragment. Furthermore, the protein was shown to react with inulin to a lesser extent. Preliminary inhibition studies revealed that sucrose but not fructose at a concentration of about 1 M was able to dissolve a levan-J606 precipitate; 0 . 2 M sucrose gave 85 per cent inhibition (Grey et al., 1971). T h e present study of the precipitin reaction pattern with different levans gives a result very similar to that previously obtained with h u m a n antilevan (Allen and Kabat 1957). I n d e e d all but one of the levan preparations react in the same order with both (Allen and Kabat, 1957). Rye grass levan is an exception in that it precipitated 1/4 of total antibody in h u m a n antilevan but negligible amounts of J606. On the other hand, inulin was a much better precipitinogen with J606 as compared with h u m a n antilevan. Inulin and rye grass levan both have molecular weights of about 5000. Rye grass levan as well as the other levan fractions are made up of /3 (2 ~ 6) fructofuranosyl residues and inulin of /3 (2 --* 1) fructofuranosyl residues. Unfortunately, however, structural data on these polysaccharides are limited. A quite different precipitin reaction pattern was obtained with a fructosan specific protein from nurse shark serum (Harisdangkul et al., 1972). With this protein inulin was f o u n d to be the best precipitinogen. Inhibition studies with J606 showed that the best inhibitors 2F1G /3DFru (2 ~ 1) /3DFru (2 -+ 6)DGlc) and 3F1G (/3DFru (2 ~ 1) /3DFru (2 ~ 1) /3DFru (2 ~ 6)DGIc) were equal on a molar basis indicating that the size of the site is

Mouse Myeioma Proteins

543

most likely that o f a trisaccharide. A f t e r reduction with NaBH4, 2F1G showed decreased activity while 3F1G similarly r e d u c e d was u n c h a n g e d . This indicates that the (2 ~ 6) linkage to the r e d u c i n g glucose unit in 2F1G has a c o n f o r m a t i o n which can fit into the site as well as does the (2 ~ 1) linkage to third fructosyl residue in 3F1G. Fructosyl sucrose (flDFru (2 ~ l)/~DFru (2 --~ I)DGIc) was a p o o r inhibitor establishing the i m p o r t a n c e o f the CH~ g r o u p next to the second fructose; ~/3(2 ~ 1) and /~(2--* 6) linked fructofuranosyl residues would be e x p e c t e d to be very similar in their three dimensional structure. T h e inhibition data differ f r o m those obtained with the fructosan specific shark protein (Harisdangkul et al., 1972) studied in this laboratory. 3F1G was considerably m o r e active than 2F1G in that system and is probably acting as a bivalent hapten. T h e shark protein displayed significant q u e n c h i n g o f fluorescence when reacting with the different fructose containing oligosaccharides used in this study. This was in sharp contrast to J606 which did not show any quenching. T h u s the changes in the two sites induced by interaction with these oligosaccharides have very different relationships to the t r y p t o p h a n e s in the two proteins. REFERENCES Allen P. Z. and Kabat E. A. (1957)J. exp. Med. 105,383. Allen P. Z. and Kabat E. A. (1959)J. Am. chem. Soc. 81, 1382. Arcamone F., Barbieri W., Cassinelli G. and Pol C. (1970) Carbohyd. Res. 14, 65. Ashman R. F. and Metzger H. (1969)J. biol. Chem. 244, 3405. BeaumontJ.°L. (1967) Compt. Rend., Acad. Sci., Set. D 264,185. Beaumont J.-L. and Lorenzelli L. (1967)Ann. Biol. Clin. 25,655. Bourne E. J., Hutson D. H. and Weigel H. (1963)Biochem.J. 86, 555. Cohn M. (1967) Cold Spring Harb. Symp. quant. Biol. 32, 211. Cohn M., Notani G. and Rice S. A. (1969) Immunoehemistry 6, 111. Eisen H. N. (1964)Meth. reed. Res. 10, 115. Eisen H. N., and Siskind G. W. (1964) Biochemistry 3,996. Eisen H. N., Little J. R., Osterland C. K. and Simms E. S. (1967) Cold Spring Harb. Symp. quant. Biol. 32, 75. Eisen H. N., Simms E. S. and Potter M. (1968) Biochemistry 7, 4126. Feizi T., Kabat E. A., Vicari G., Anderson B. and Marsh W. L. (1971)J. lmmun. 106, 1578. GoodmanJ. W. and Kabat E. A. (1964)J. Immun. 93,213. Grey H. M., HirstJ. W. and Cohn M. (1971)J. exp. Med. 133,289. Harboe M. (1965) Ser. Haematol. 4, 67. Harisdangkul V., Kabat E. A. and McDonough R. and Sigel M. M. (1972)J. Immun. (in press). Hehre E.J. and SuggJ. Y. (1942)J. exp. Med. 75,339. Jaffe B. M., Eisen H. N., Simms E. S. and Potter M. (1969)J. Immun. 103, 879. Jaffe B. M., Simms E. S. and Eisen H. N. (1971) Biochemistry 10, 1693. Johnston I. R. (1965) Biochem.J., 96, 659. Kabat E. A. (1961) Kabat and Mayer's Experimental lmmunochemistry, 2nd Edition. Thomas, Springfield, I11. Leon M. A., Young N. M. and McIntire K. R. (1970)Biochemistry9, 1023. Mage R. G. and Kabat E. A. (1963)J. Immun. 91,633. Metzger H. (1967) Proc. natn. Acad. Sci. U.S.A. 57, 1490. NeillJ. M., Hehre E.J., SuggJ. Y. and Jaffe E. (1939)J. exp. Med. 70,427. NeillJ. M., SuggJ. Y. and Jaffe E. (1941) Am.J. Hyg. 34, 65. Potter M. and Leon M. (1968) Science 162,369. Potter M. and Lieberman R. (1970)J. exp. Med. 132, 737. Potter M. (1970) Fedn Proc. 29, 85.

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Schiffman G., Kabat E. A. and Thompson W. (1964) Biochemistry 3, 113. Schubert D.,Jobe A. and Cohn M. (1968) Nature, Lond. 220, 882. Schubert D., Roman A. and Cohn M. (1970) Nature, Lond. 225, 154. Stone M. J. and Metzger H. (1969)J. lmmun. 102,222. Terry W. D., Ashman R. F. and Metzger H. (1970a) Immunochemistry 7, 257. Terry W. D., Boyd M. M., ReaJ. S. and Stein R. (1970b)J. Immun. 104, 256. Torii M., Kabat E. A. and Weigel H. (1966)J. Immun. 96, 797. Tung K. K. and NordinJ. H. (1970)Analyt. Biochem. 38, 164. Velick S. F., Parker C. W. and Eisen H. N. (1960) Proc. natn. Acad. Sci. U.S.A. 46, 1470. Vicari G., Sher A., Cohn M. and Kabat E. A. (1970) Immunochemistry 7, 829. Warner N. L. and Ovary Z. (1970)J. Immun. 105, 812. Weigert M. G., Cesari I. M., Yonkovich S. J., and Cohn M. (1970) Nature, Lond. 228, 1045. Zettervall O. (1967)Acta Med. Scand. Suppl. 49,215.