Degradation of a Pneumococcal Type-Specific Polysaccharide with Exposure of Group-Specificity*

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Proceedings of the National Academy of Sciences Vol. 67, No. 1, pp. 138-142, September 1970 Degradation of a Pneumococcal Type-Specific Polysaccharide with Exposure of Group-Specificity* John D.

1 Proceedings of the National Academy of Sciences Vol. 67, No. 1, pp , September 1970 Degradation of a Pneumococcal Type-Specific Polysaccharide with Exposure of Group-Specificity* John D. Higginbotham, Michael Heidelberger, and Emil C. Gotschlicht DEPARTMENT OF PATHOLOGY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE, NEW YORK 10016; AND tthe ROCKEFELLER UNIVERSITY, NEW YORK Communicated July 1, 1970 Abstract. Pyruvic acid is an immunological determinant of the quite rigorously type-specific capsular polysaccharide of pneumococcal type IV (S IV). Removal of pyruvic acid by mild hydrolysis converts the capsular polysaccharide of type IV into an analog of the pneumococcal group-specific C-substance. Depyruvylated S IV resembles C-substance so closely immunologically that it not only precipitates a high proportion of the anti-c in antipneumococcal sera, regardless of their immunological types, but also, like C, precipitates human C-reactive protein in the presence of calcium ions. Apparently, the removal of pyruvic acid ketal rings from adjacent sugars unmasks N-acetylgalactosamine residues which must be linked and spaced much as are those in C-substance. Groupings reactive with suitably linked N-acetylgalactosamine, therefore, appear to be located on the surfaces of molecules of human C-reactive protein. Pyruvic acid has been found, from time to time, in bacterial polysaccharides but its immunological importance has only recently been realized.1 Linked to two hydroxyls of a sugar residue as a ketal2'3 it becomes a factor in the serological reactions of microorganisms as widely disparate as Rhizobia, pneumococci, and Klebsiella.4 We have therefore resumed the study of the capsular polysaccharide (S IV) of pneumococcal type IV,5 which contains pyruvic acid. Its components are, in addition, D-galactose and three amino sugars, galactosamine, mannosamine, and another, probably fucosamine, presumably present as their N-acetyl derivatives. The present report covers the removal of pyruvic acid from S IV and two unusual immunological consequences of its cleavage. Materials and Methods. S IV was prepared by Dr. Paul A. Rebers in 1958 at the Institute of Microbiology, Rutgers, The State University, New Brunswick, N.J., much as in an earlier study.6 The type IV pneumococci were grown at Fort Detrick, Md., courtesy of Dr. Riley D. Housewright, and were killed with phenol before collection. Antipneumococcal (anti-pn) horse sera were obtained from the Laboratories of the New York City and New York State Departments of Health through Drs. Paul S. May and Kenneth Amiraian. Use was also made of a rabbit antiserum to a rough strain of pneumococcus. Studies of the reaction of the rabbit antiserum with C polysaccharide and the inhibition of this reaction by sugars have been published.7 Analyses for the amounts of antibody nitrogen precipitated, in homologous and crossreactions in antipneumococcal sera, were carried out as in earlier papers.8'9 138

2 VOL. 67, 1970 DEGRADATION OF PNEUMOCOCCAL POLYSACCHARIDE 139 Crystalline human C-reactive protein was prepared as previously described.'0 Its precipitation by the polysaccharides was measured quantitatively by solution of the precipitates in 1.0 ml of 0.1 N NaOH and estimation of the protein by a modification of the Folin reaction,1' with the use of a Technicon auto-analyzer. Pyruvic acid was estimated as in reference 12 but on a smaller scale, choline as in reference 13. Results and Discussion. S IV contains 8-9% of pyruvic acid. The carboxyl group is titratable after passage of S IV through a Bio-Rad AG50W X 8(H+) column; found, 5.1% COOH; calcd. for 8.5% pyruvic acid, 4.4% COOH. The kinetics of the removal of pyruvic acid from S IV at 100'C, in 0.01 N HCl, are shown in Figure 1. Cleavage of pyruvic acid from the residual polysaccharide is W 6 FIG. 1.-Rate of release of 5 pyruvic acid from S IV in 0.01 N 4 HC1 at 1000C. ar 3 ar2 / ' E TIME (MINUTES) almost complete in 6 min, and is accompanied by removal of sugars only on longer heating. Pyruvic acid is eliminated much more slowly from S XXVII, the only other pneumococcal polysaccharide thus far reported to contain the acid." 4 Pn C, S IX, S XII, and S XVI gave negative tests. Profound changes occur in the immunological specificity of S IV when pyruvic acid is split off to form the depyruvylated derivative, dps IV. Liberation of two additional hydroxyl groups of a sugar (probably D-galactose), comprising a portion of the presumed repeating unit, is accompanied by the appearance of crossreactions not shown by the intact substance. This also occurred among the rhizobial polysaccharides studied.4 The most striking and unusual change, however, was the conversion of S IV, one of the most rigorously type-specific of the pneumococcal polysaccharides, into a substance with pronounced group-specificity. As shown in Table 1, dps IV precipitates a very large proportion of antibodies to the C-polysaccharide, the major group-specific antigen of pneumococci. Antisera raised in horses to types I, VII, XII, and XXVIII were chosen because they were the richest in anti-c of those at hand. Comparison is also made with a non-type-specific rabbit anti-c. The group-specific pneumococcal C-substance, first described by Tillett and Francis,'4 was shown to contain bound phosphorus5 and, more recently, to consist of N-acetylgalactosamine-6-phosphate,7 choline,'5"6 glucose, 2-acetamino- 4-amino-2,4,6-trideoxyhexose, and amino acids. The dps IV used in most

140 IMMUNOLOGY: HIGGINBOTHAM ET AL. PROC. N. A. S. TABLE 1. Maximal cross-reactivity of S IV and dps IV with antibodies to pneumococcal C-polysaccharide. Amount (l.

3 140 IMMUNOLOGY: HIGGINBOTHAM ET AL. PROC. N. A. S. TABLE 1. Maximal cross-reactivity of S IV and dps IV with antibodies to pneumococcal C-polysaccharide. Amount (l.g) of antibody nitrogen precipitated at OC, calculated to 1.0 ml. Anti-Pa, horse, type: Polysaccharide I1057 I1057Cb VII1074 VII1074Cb Pn Ca S IV 10 0 dp S IV 269c d 5 Anti-Pn, horse, type-- Anti-Pn R36A, Polysaccharide X11625 XII625Cb XXVIII510 rabbit 1238 Pn Ca S IV dp S IV 334 trace a Different preparations of C-polysaccharide precipitated somewhat different amounts of N. b Absorbed with Pn C. c Supernatants gave 27 ug N with Pn C. d Supernatants gave 14 pg N with Pn C. of our experiments contained a small amount of C-substance, 0.4%o P, and 0.9% choline, but it was almost as effective, per unit of weight, as C in the precipitation of anti-c. Fractionation of S IV on DEAE-Sephadex gave a small portion free of C-substance, as judged by failure to show a line on gel-diffusion with anti- C, and by absence of choline after depyruvylation. This portion of purified dps IV gave the same reactions as the other sample. Hence, the acquisition of C-like specificity was not due to contaminating C-substance. Gotschlich and Liu have shown7 that N-acetylgalactosamine-6-phosphate is the immunodominant sugar of the pneumococcal C-polysaccharide. Attribution of this property to choline18 appears to be without published foundation, since reference is made to cross-reactions of C with myeloma proteins. Since N- acetylgalactosamine is the only substance common to S IV and C-polysaccharide, this sugar would appear to be blocked to the approach of antibody in intact S IV by the ring CH3I ACOOH l 2 formed by the ketal linkage to two hydroxyl groups of an adjacent sugar, presumably D-galactose. After removal of the pyruvate, the multiple residues (cf. reference 19) of N-acetylgalactosamine evidently become exposed and accessible to anti-c, and must be oriented in space in a fashion extraordinarily similar to many of those in the C-polysaccharide itself, since so high a proportion of the anti-c is precipitated. This close immunological correspondence in dps IV is also demonstrated by a second new property; namely, precipitation with C-reactive protein, a substance which appears in numerous pathological conditions. The amount of C-reactive protein precipitated by dps IV and C polysaccharide is nearly the same, although approximately 8 times as much dps IV as C-polysaccharide is required. Intact S IV reacts negligibly. As with C-polysaccharide, the reaction with dps IV is

VOL. 67, 1970 DEGRADATION OF PNEUMOCOCCAL POLYSACCHARIDE 141 TABLE 2. Precipitation of C-reactive protein by Pn C, S IV, and dps IV. jug protein precipitated by jg antigen used Pn C dps IV S IV 1 14.

4 VOL. 67, 1970 DEGRADATION OF PNEUMOCOCCAL POLYSACCHARIDE 141 TABLE 2. Precipitation of C-reactive protein by Pn C, S IV, and dps IV. jug protein precipitated by jg antigen used Pn C dps IV S IV a a 128 jig of C-free dps IV precipitated 142 ug of C-reactive protein. inhibited by chelation of divalent cations with sodium EDTA. Until now it was not established which determinant in C-polysaccharide accounts for its reaction with C-reactive protein. The prime suspects were N-acetylgalactosamine or its phosphorylated derivative, choline, or a 2-acetamino-4-amino-2,4,6-trideoxyhexose. Since choline-free dps IV precipitates C-reactive protein, and since S IV and dps IV are stable to nitrous acid and so do not contain 2-acetamino-4- aminotrideoxyhexose in a linkage similar to that in C-substance, it would appear that C-reactive protein carries receptor groupings for N-acetylgalactosamine in suitable linkage. It is now known that the numerous type-specificities of the "O" lipopolysaccharides of Salmonella are a consequence of side-chains grafted upon a common group-specific core (reviews, reference 20). This is, however, the first time, to our knowledge, that a similar, albeit much simpler, instance has been shown among pneumococci. Chemical studies on S IV are continuing and will be reported separately. Just how dps IV is related to the C-like C8 capsular polysaccharide2l must await the promised chemical studies on the latter. The technical assistance of W. P. Grosvenor is gratefully acknowledged. Abbreviations: S IV, type-specific capsular polysaccharide of pneumococcal type IV; dps IV, the depyruvylated derivative of S IV; anti-pn, anti-pneumococcal; Pn C, pneumococcal C polysaccharide. * Carried out with the aid of Grants GB from the National Science Foundation, and HE from the U.S. Public Health Service, respectively. 'Dudman, W. F., and M. Heidelberger, Science, 164, 954 (1969). 2Orentas, D. G., J. H. Sloneker, and A. Jeanes, Can. J. Microbiol., 9, 427 (1963); Gorin, P. A. J., T. Ishikawa, J. F. T. Spencer, and J. H. Sloneker, Can. J. Chem., 45, 2005 (1967). 8 Hirase, S., Bull. Chem. Soc. Jap., 30, 70 (1957); Gorin, P. A. J., and T. Ishikawa, Can. J. Chem., 45, 521 (1967). 4Heidelberger, M., W. F. Dudman, and W. Nimmich, J. Immunol., 104, 1321 (1970). 6 Heidelberger, M., and F. E. Kendall, J. Exp. Med., 53, 625 (1931). 6 Heidelberger, M., C. M. MacLeod, H. Markowitz, and A. S. Roe, J. Exp. Med., 91, 341 (1950). 7Gotschlich, E. C., and T. Y. Liu, J. Biol. Chem., 242, 463 (1967). 8 Heidelberger, M., and P. A. Rebers, J. Amer. Chem. Soc., 80, 116 (1958). 9 Heidelberger, M., H. Jahrmarker, B. Bjorklund, and J. Adams, J. Immunol., 78, 419 (1957). "0 Gotschlich, E. C., and G. M. Edelman, Proc. Nat. Acad. Sci. USA, 54, 558 (1965).

5 142 IMMUNOLOGY: HIGGINBOTHAM ET AL. PROC. N. A. S. 11 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 12 Sloneker, J. H., and D. G. Orentas, Nature, 194, 478 (1962). 13 Gourley, W. K., C. D. Haas, and S. Bakerman, Anal. Biochem., 19, 197 (1967). 14 Tillett, W. S., and T. Francis, Jr., J. Exp. Med., 52, 561 (1930). 15 Tomasz, A., Science, 157, 694 (1967); Mosser, J. L. and A. Tomasz, J. Biol. Chem., 245, 287 (1970). 16 Brundish, D. E., and J. Baddiley, Biochem. J., 105, 30c (1967); 110, 573 (1968). 17 Distler, J., B. Kaufman, and S. Roseman, Arch. Biochem. Biophys., 116, 466 (1966). 18 Potter, M., R. Lieberman, L. Hood, and D. J. McKean, Fed. Proc., 29, abstr (1970). 19 Heidelberger, M., and F. E. Kendall, J. Exp. Med., 61, 563 (1935). 20 Luderitz, O., A. M. Staub, and 0. Westphal, Bacteriol. Rev., 30, 192 (1966); Nikaido, H., Advan. Enzymol. Relat. Areas Mol. Biol., 31, 77 (1968). 21 Bornstein, D. L., G. Schiffman, H. P. Bernheimer, and R. Austrian, J. Exp. Med., 128, 1385 (1968).

CHEMICAL STUDIES ON BACTERIAL AGGLUTINATION II. THE IDENTITY OF PRECIPITIN AND AGGLUTININ* BY MICHAEL HEIDELBERGER, PH.D., AND ELVIN A.

CHEMICAL STUDIES ON BACTERIAL AGGLUTINATION II. THE IDENTITY OF PRECIPITIN AND AGGLUTININ* BY MICHAEL HEIDELBERGER, PH.D., AND ELVIN A. KABAT (From the Laboratories of the Departments of Medicine and Biological