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Immunochemical properties of oligosaccharide-protein conjugates withKlebsiella-K2 specificity

I. Specificity and crossreactivity of anti-conjugate versus anti-bacterial antibodies

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Abstract

A series of repeating unit oligomers

$$\begin{gathered} \alpha - GlcUA \hfill \\ 1 \downarrow 3 \hfill \\ [ \to 4) - \beta - Man - (1 \to 4) - \alpha - Glc - (1 \to 3) - \beta - Glc - (1 \to ]_n , 1 \leqslant n \leqslant 7 , \hfill \\ \end{gathered} $$

was prepared by depolymerization ofKlebsiella pneumoniae B5055 (01∶K2) capsular polysaccharide (K2-PS) as catalyzed by a bacteriophage-associated glycanase. The monomeric repeating unit [tetrasaccharide, (TS)] and its di- and trimer [octa- and dodecasaccharide (OS and DS)] were conjugated to edestin via reductive aminophenylation and azo coupling at the reducing sugar end group. Rabbit anti-conjugate and anti-bacterial antibodies raised in rabbits were compared with respect to specificity and crossreactivity towards the oligomers of the various molecular sizes, towards parent PS, and towards whole bacteria. Antibodies able to bind specifically to the PS and the bacteria were elicited by all three conjugates. However, the anti-TS conjugate antibodies, in contrast to those obtained with OS and DS conjugate, proved to be practically unable to effect bacterial agglutination. Correspondingly, the TS played an exceptional role in binding to anti-bacterial antibodies. In contrast to the OS and DS it could not fully inhibit precipitation of these antibodies with the bacterial PS. Moreover, the inhibition of the binding of the PS to antibacterial antibodies produced by TS was about 50-fold weaker than that produced by OS, DS, and higher members of the series, all of which were about equally potent inhibitors (on a molar basis). The results show that two repeating units are the minimum requirement for a substantial representation of the PS's serologic specificity. The exceptional behavior of the TS correlates with its lack of theβ-Glc-(1–4)-Man linkage present in all higher members of the series.

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Abbreviations

PBS:

phosphate-buffered saline

PFU:

plaque-forming units

PS:

polysaccharide

K2-PS:

Klebsiella pneumoniae B5055 (01∶K2) capsular polysaccharide

TS:

tetrasaccharide (=monomeric repeating unit)

OS:

octasaccharide (=dimeric repeating unit)

DS:

dodecasaccharide (=trimeric repeating unit)

NaBH3CN:

sodium cyanoborohydride

HHG:

human γ-globulin

FCA:

Freund's complete adjuvant

i.m.:

intramuscularly

i.p.:

intraperitoneally

TS125I, PS125I:

iodine-125-labelled TS or K2-PS, respectively

References

  1. Arakatsu, Y., Ashwell, G., Kabat, E.A.: Immunochemical studies on dextrans. V. Specificity and crossreactivity with dextrans of the antibodies formed in rabbits to isomaltonic and isomaltotrionic acids coupled to bovine serum albumin. J. Immunol.97, 858–866 (1966)

    PubMed  Google Scholar 

  2. Avery, O.T., Goebel, W.F.: Chemo-immunological studies on conjugated carbohydrate-proteins. II. Immunological specificity of synthetic sugar-protein antigens. J. Exp. Med.50, 533–550 (1929)

    Article  Google Scholar 

  3. Avrameas, S., Ternyck, T.: The cross-linking of protein with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry6, 53–66 (1969)

    Article  PubMed  Google Scholar 

  4. Axen, R., Porath, J., Ernback, S.: Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature214, 1302–1305 (1967)

    PubMed  Google Scholar 

  5. Bitter, T., Muir, H.M.: A modified uronic acid carbazole reaction. Anal. Biochem.4, 330–334 (1962)

    Article  PubMed  Google Scholar 

  6. Bjömdal, H., Hellerqvist, C.G., Lindberg, B., Svensson, S.: Gas-Flüssigkeits-Chromatographie und Massenspektrometrie bei der Methylierungsanalyse von Polysacchariden. Angew. Chemie82, 643–674 (1970)

    Google Scholar 

  7. Cheng, F.W., Cannon, L.E., Margolies, M.N., Strosberg, A.D., Haber, E.: Purification, specificity, and hypervariable region sequence of anti-pneumococcal polysaccharide antibodies elicited in a single rabbit. J. Immunol.117, 807–813 (1976)

    PubMed  Google Scholar 

  8. Coligan, J.E., Fraser, B.A., Kindt, T.J.: A disaccharide hapten from streptococcal group C carbohydrate that crossreacts with the Forssman glycolipid. J. Immunol.118, 6–11 (1977)

    PubMed  Google Scholar 

  9. Crothers, D.M., Metzger, H.: The influence of polyvalency on the binding properties of antibodies. Immunochemistry9, 341–357 (1972)

    Article  PubMed  Google Scholar 

  10. Cuatrecasas, P., Anfisen, C.B.: Affinity chromatography. Ann. Rev. Biochem.40, 259–277 (1971)

    Article  PubMed  Google Scholar 

  11. Edwards, P.R., Ewing, W.H. (eds.): Identification ofEnterobacteriaceae: Minneapolis: Burgess 1966

    Google Scholar 

  12. Gahan, L.C., Sandford, P.A., Conrad, H.E.: The structure of the serotype 2 capsular polysaccharide ofAerobacter aerogenes. Biochemistry6, 2755–2767 (1967)

    Article  PubMed  Google Scholar 

  13. Galanos, C., Lüderitz, O., Himmelspach, K.: The partial acid hydrolysis of polysaccharides: A new method for obtaining oligosaccharides in high yield. Eur. J. Biochem.8, 332–336 (1969)

    Article  PubMed  Google Scholar 

  14. Geyer, H., Schlecht, S., Himmelspach, K.: In preparation

  15. Goebel, W.F., Avery, O.T.: Chemo-immunological studies on conjugated carbohydrate-proteins. I. The synthesis of p-aminophenolβ-glucoside, p-aminophenolβ-galactoside and their coupling with serum globulin. J. Exp. Med.50, 521–531 (1929)

    Article  Google Scholar 

  16. Goebel, W.F.: Chemo-immunological studies on conjugated carbohydrate-proteins. XII. The immunological properties of an artificial antigen containing cellobiuronic acid. J. Exp. Med.68, 469–484 (1938)

    Article  Google Scholar 

  17. Goebel, W.F.: Antibacterial immunity induced by artificial antigens. I. Immunity of experimental pneumococcal infection with an antigen containing cellobiuronic acid. J. Exp. Med.69, 353–364 (1939)

    Article  Google Scholar 

  18. Hämmerling, U.: Immunchemische Untersuchungen zur Überführung von bakteriellen Polysaccharid-Haptenen in Vollantigene. Dissertation Universität Freiburg (1965)

  19. Hakomori, S.: A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. J. Biochem.55, 205–207 (1964)

    PubMed  Google Scholar 

  20. Hellerqvist, C.G., Lindberg, B., Svensson, S., Holme, T., Lindberg, A.A.: Structural studies on the O-specific side-chains of the cell-wall lipopolysaccharide fromSalmonella typhimurium 395MS. Carbohydr. Res.8, 43–55 (1968)

    Article  Google Scholar 

  21. Himmelspach, K., Westphal, O., Teichmann, B.: Use of 1-(m-aminophenyl) flavazoles for the preparation of immunogens with oligosaccharide determinant groups. Eur. J. Immunol.1, 106–112 (1971)

    PubMed  Google Scholar 

  22. Himmelspach, K., Geyer, H., Hoyer, G., Schepers, G.: Preparation of125I-labelled oligosaccharide derivatives with the aid of 3-(4-hydroxyphenyl)propionic acid N-hydroxy-succinimide ester. FEBS Letters75, 154–158 (1977)

    Article  PubMed  Google Scholar 

  23. Hornick, C.L., Karush, F.: Antibody affinity-III. The role of multivalence. Immunochemistry9, 325–340 (1972)

    Article  PubMed  Google Scholar 

  24. Howard, J.G., Vicary, G., Courtenay, B.M.: Influence of molecular structure on the tolerogenicity of bacterial dextrans. I. Theα 1-6-linked epitope of dextran B5 12. Immunology29, 585–597 (1975)

    PubMed  Google Scholar 

  25. Hunter, W.M., Greenwood, F.C.: Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature194, 495–496 (1962)

    PubMed  Google Scholar 

  26. Imoto, T., Yagishita, K.: A simple activity measurement of lysozyme. Agr. Biol. Chem.35, 1154–1156 (1971)

    Google Scholar 

  27. Jann, K., Jann, B., Ørskov, F., Ørskov, I., Westphal, O.: Immunchemische Untersuchungen an K-Antigenen vonEscherichia coli. II. Das K-Antigen von E.coli 08∶K42(A)∶H−. Biochem. Z.342, 1–22 (1965)

    PubMed  Google Scholar 

  28. Jann, K., Westphal, O.: Microbial polysaccharides. In: The Antigens (Sela, M., ed.) Vol. 3, pp. 1–125. New York: Academic Press 1975

    Google Scholar 

  29. Jeffrey, A.M., Zopf, D.A., Ginsburg, V.: Affinity-chromatography of carbohydrate-specific immunoglobulins: Coupling of oligosaccharides to Sepharose. Biochem. Biophys. Res. Commun.62, 608 (1974)

    Article  Google Scholar 

  30. Kabat, E.A., Mayer, M.M.: Experimental Immunochemistry, 2nd edition, pp. 241–264. Springfield: C.C. Thomas 1961

    Google Scholar 

  31. Kamicker, B.J., Schwartz, B.A., Olson, R.M., Drinkwitz, D.C., Gray, G.R.: Proteins containing reductively aminated disaccharides: Immunochemical characterization. Arch. Biochem. Biophys.183, 393–398 (1977)

    Article  PubMed  Google Scholar 

  32. Kauffmann, F.: The bacteriology ofEnterobacteriaceae, Kopenhagen: Munksgaard 1966

    Google Scholar 

  33. Kickhöfen, B., Warth, R.: Eine Trennkammer für die Hochspannungselektrophorese nach dem Michl'schen Prinzip. J. Chromatogr.33, 558–560 (1968)

    Article  Google Scholar 

  34. Kim, Y.T., Kalver, S., Siskind, G.W.: A comparison of the Farr technique with equilibrium dialysis for measurement of antibody concentration and affinity. J. Immunol. Meth.6, 347–354 (1975)

    Article  Google Scholar 

  35. Kleinhammer, G., Himmelspach, K., Westphal, O.: Synthesis and immunological properties of an artificial antigen with the repeating oligosaccharide unit ofSalmonella illinois as haptenic group. Eur. J. Immunol.3, 834–838 (1973)

    PubMed  Google Scholar 

  36. Kochetkov, N.K., Dimitriev, B.A., Chizhov, O.S., Klimov, E.M., Malysheva, N.N., Chernyak, A.Ya., Bayramova, N.E., Torgov, V.I.: Synthesis of derivatives of the trisaccharide repeating unit of the O-specific polysaccharide fromSalmonella anatum. Carbohydr. Res.33, C5-C7 (1974)

    Article  Google Scholar 

  37. Krebs, K.G., Heusser, D., Wimmer, H.: Sprühreagentien. In: Dünnschichtchromatographie (Stahl, E., ed.). pp. 813–861. Berlin: Springer 1967

    Google Scholar 

  38. Lemieux, R.U., Bundle, D.R., Baker, D.A.: The properties of a “synthetic” antigen related to the human blood-group Lewis a. J. Amer. Chem. Soc.97, 4076–4083 (1975)

    Article  Google Scholar 

  39. Lindberg, B.: Methylation analysis of polysaccharides. In: Methods in Enzymology, Vol. XXVIII: Complex Carbohydrates, Part B (Ginsburg, V., ed.). pp. 178–195. New York: Academic Press 1972

    Google Scholar 

  40. Lönngren, J., Goldstein, I.J., Niederhuber, J.E.: Aldonate Coupling, a simple procedure for the preparation of carbohydrate-protein conjugates for studies of carbohydrate-binding proteins. Arch. Biochem. Biophys.175, 661–669 (1976)

    Article  PubMed  Google Scholar 

  41. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem.193, 265–275 (1951)

    PubMed  Google Scholar 

  42. McBroom, C.R., Samanen, C.H., Goldstein, I.J.: Carbohydrate antigens: Coupling of carbohydrates to proteins by diazonium and phenylisothiocyanate reactions. In: Methods in Enzymology, Vol. XXVIII: Complex Carbohydrates, Part B (Ginsburg, V., ed.), pp. 212–219. New York: Academic Press 1972

    Google Scholar 

  43. Meyer-Delius, M., Mitchinson, N.A., Pitt-Rivers, R., Rüde, E.: Synthesis of a radioiodine-labelled 2,4-dinitrophenyl-hapten and its use for binding assays. Eur. J. Immunol.1, 267–271 (1971)

    PubMed  Google Scholar 

  44. Minden, P., Farr, R.S.: In: Handbook of experimental immunology (Weir, D.M., ed.). p. 465. Oxford: Blackwell Scientific Publications (1967)

    Google Scholar 

  45. Morrison, J.M.: Determination of the degree of polymerization of oligo- and polysaccharides by gas-liquid chromatography. J. Chromatogr.108, 361–364 (1975)

    Article  PubMed  Google Scholar 

  46. Niemann, H., Beilharz, H., Stirm, S.: Bacteriophage-borne enzymes in carbohydrate chemistry. II. Kinetics and substrate specificity of the glycanase activity associated with particles of theKlebsiella bacteriophage No.13. Carbohydr. Res.60, 353–366 (1978)

    Google Scholar 

  47. Nimmich, W.: Zur Isolierung und qualitativen Bausteinanalyse der K-Antigene von Klebsiellen. Z. Med. Mikrobiol. Immunol.154, 117–131 (1968)

    Article  PubMed  Google Scholar 

  48. Osborn, M.J.: Studies on the gram-negative cell wall. I. Evidence for the role of 2-keto-3-deoxyoctonate in the lipopolysaccharide ofSalmonella typhimurium. Proc. Natl. Acad. Sci., USA50, 499–506 (1963)

    Google Scholar 

  49. Ottenstein, D.M., Bartley, D.A.: Separation of free acids C2-C5 in dilute aqueous solution column technology. J. Chromatogr. Sci.9, 673–681 (1971)

    Google Scholar 

  50. Porath, J., Asperg, K., Drevy, H., Axen, R.: Preparation of cyanogen bromide-activated agarose gels. J. Chromatogr.86, 53–56 (1973)

    Article  Google Scholar 

  51. Sawardeker, J.S., Sloneker, J.H., Jeanes, A.: Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal. Chem.12, 1602–1604 (1965)

    Article  Google Scholar 

  52. Schlecht, S., Westphal, O.: Wachstum und Lipopolysaccharid (O-Antigen)-Gehalt von Salmonellen bei Züchtung aus Agarnährböden. Zbl. Bakteriol. Parasitenk. Abt. I, Orig. A.,200, 241–259 (1966)

    Google Scholar 

  53. Seppälä, I.J.T.: Disturbance of hapten-antibody equilibria by ammonium sulphate solutions. A source of error in antibody affinity determinations. J. Immunol. Meth.9, 135–140 (1975)

    Article  Google Scholar 

  54. Shields, R., Burnett, W.: Determination of protein-bound carbohydrate in serum by a modified anthrone method. Analyt. Chem.32, 885–886 (1960)

    Article  Google Scholar 

  55. Stirm, S., Freund-Mölbert, E.:Escherichia coli capsule bacteriophages. II. Morphology. J. Virol.8, 330–342 (1971)

    PubMed  Google Scholar 

  56. Sutherland, I.W.: The exopolysaccharides ofKlebsiella serotype 2 strains as substrates for phage-induced polysaccharide depolymerases. J. Gen. Microbiol.70, 331–338 (1971)

    Google Scholar 

  57. Thurow, H., Niemann, H., Rudolph, C., Stirm, S.: Host capsule depolymerase activity of bacteriophage particles active onKlebsiella K20 and K24 strains. Virology58, 306–309 (1974)

    Article  PubMed  Google Scholar 

  58. Thurow, H., Choy, Y.M., Frank, N., Niemann, H., Stirm, S.: The structure ofKlebsiella serotype 11 capsular polysaccharide. Carbohydr. Res.41, 241–255 (1975)

    Article  PubMed  Google Scholar 

  59. Thurow, H., Niemann, H., Stirm, S.: Bacteriophage-borne enzymes in carbohydrate chemistry. Part I. On the glycanase activity associated with particles ofKlebsiella bacteriophage No. 11. Carbohydr. Res.41, 257–271 (1975)

    Article  PubMed  Google Scholar 

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  1. Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-7800, Freiburg/Br., Federal Republic of Germany

    Hildegard Geyer, Stephan Stirm & Karl Himmelspach

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  1. Hildegard Geyer
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  2. Stephan Stirm
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Geyer, H., Stirm, S. & Himmelspach, K. Immunochemical properties of oligosaccharide-protein conjugates withKlebsiella-K2 specificity. Med Microbiol Immunol 165, 271–288 (1979). https://doi.org/10.1007/BF02152925

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