A novel strategy for the synthesis of neoglycoconjugates from deacylated deep rough lipopolysaccharides

Abstract

We report a novel strategy for the preparation of neoglycoconjugates of oligosaccharides which are obtained after complete deacylation of bacterial deep rough lipopolysaccharides (LPS) isolated from recombinant Escherichia coli bacteria synthesizing a Kdo di-[α-Kdo-(2→4)-α-Kdo-(2→] and a Kdo trisaccharide [α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo-(2→] of Re-type and chlamydial LPS, respectively. Unlike acylated LPS, such oligosaccharides can be obtained in pure form and thus lead to well-defined neoglycoconjugates. Cleavage of the 1-phosphate of the lipid A moiety by alkaline phosphatase treatment leads to a free reducing glucosamine which can be further reacted with allylamine. After reductive amination, spacer elongation of the allyl group with cysteamine and activation with thiophosgene, the ligands were reacted with BSA. We have compared the immunological reactivity of such defined neoglycoconjugates obtained from natural sources with those obtained by chemical synthesis and report that such neoglycoconjugates are immunogenic and well suited as antigens for the study of epitope specificities of monoclonal antibodies. In addition, we have compared these conjugates with those in which ligands were coupled by glutardialdehyde to BSA. Our approach proved to be superior since the latter led upon immunization of mice to a relatively high percentage of antibodies that reacted with glutardialdehyde derivatized BSA without the carbohydrate ligand. This was not the case for cysteamine-spacered ligands coupled via their isothiocyanate-derivatives.

References

Mamat U. , Seydel U. , Grimmecke D. , Holst O. , Rietschel ET Lipopolysaccharides, In: Pinto BM. (ed) Carbohydrates and their Derivatives including Tannins, Cellulose, and Related Lignins. Amsterdam: Elsevier, 1999; 179—239.

Müller-Loennies S. , Di Padova FE, Brade L, Heumann D, Rietschel ET. Conserved epitopes in bacterial lipopolysaccharides and cross-reactive antibodies in the treatment of endotoxicosis and Gram-negative septic shock. In: Andrew PW , Oyston P , Smith GL , Stewart-Tull DE. (eds) Fighting Infection in the 21st Century. Oxford: Blackwell, 2000; 143—178.

Grimmecke HD , Brade H. Studies on the reductive amination of 3-deoxy-D-manno-octulosonic acid (Kdo). Glycoconj J 1998; 15: 555—562.

Holst O. , Broer W. , Thomas-Oates JE , Mamat U. , Brade H. Structural analysis of two oligosaccharide bisphosphates isolated from the lipopolysaccharide of a recombinant strain of Escherichia coli F515 (Re chemotype) expressing the genus-specific epitope of Chlamydia lipopolysaccharide. Eur J Biochem 1993; 214: 703—710.

Müller-Loennies S. , Holst O. , Lindner B. , Brade H. Isolation and structural analysis ofphosphorylatedoligosaccharides obtained from Escherichia coli J-5 lipopolysaccharide. Eur J Biochem 1999; 260: 235—249.

Holst O. , Thomas-Oates JE , Brade H. Preparation and structural analysis of oligosaccharide monophosphates obtained from the lipopolysaccharide of recombinant strains of Salmonella minnesota and Escherichia coli expressing the genus-specific epitope of Chlamydia lipopolysaccharide . Eur J Biochem 1994; 222: 183—194.

Galanos C. , Lüderitz O. , Westphal O. A new method for the extraction of R lipopolysaccharides. Eur J Biochem 1969; 9: 245—249.

Kosma P. , Schulz G. , Brade H. Synthesis of a trisaccharide of 3deoxy-D-manno-2-octulopyranosylonic acid (KDO) residues related to the genus-specific lipopolysaccharide epitope of Chlamydia. Carbohydr Res 1988; 183: 183—199.

Lee RT , Lee YC Synthesis of 3-(2-aminoethylthio)propyl glycosides. Carbohydr Res 1974; 37: 193—201.

10.

Fu Y. , Baumann M. , Kosma P. , Brade L. , Brade H. A synthetic glycoconjugate representing the genus-specific epitope of chlamydial lipopolysaccharideexhibits the same specificity as its natural counterpart . Infect Immun 1992; 60: 1314—1321.

11.

Brade L. , Brunnemann H. , Ernst M. et al. Occurrence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen . FEMS Immunol Med Microbiol 1994; 8: 27—41.

12.

Brade L. , Grimmecke HD , Holst O. , Brabetz W. , Zamojski A. , Brade H. Specificity of monoclonal antibodies against Escherichia coli K- 12 lipopolysaccharide . J Endotoxin Res 1996; 3: 39—47.

13.

Rozalski A. , Brade L. , Kosma P. , Appelmelk BJ , Krogmann C. , Brade H. Epitope specificities of murine monoclonal and rabbit polyclonal antibodies against enterobacterial lipopolysaccharides of the Re chemotype. Infect Immun 1989; 57: 2645—2652.

14.

Kosma P. , Reiter A. , Hofinger A. , Brade L. , Brade H. Synthesis of neoglycoproteins containing Kdo epitopes specific for Chlamydophila psittaci lipopolysaccharide. J Endotoxin Res 2000; 6: 57—69.

15.

Maaheimo H. , Kosma P. , Brade L. , Brade H. , Peters T. Mapping the binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharideto antibodies with NMR . Biochemistry 2000; 39: 12778—12788.

16.

Brade L. , Zych K. , Rozalski A. , Kosma P. , Bock K. , Brade H. Structural requirements of synthetic oligosaccharides to bind monoclonal antibodies against Chlamydia lipopolysaccharide. Glycobiology 1997; 7: 819—827.

17.

LikhosherstovLM, NovikovaOS , Derevitskaja VA , Kochetkov NK A new simple synthesis of amino sugars β-D-glycosyl-amines. Carbohydr Res 1986; 146: C1—C5.

18.

Manger ID , Rademacher TW , Dwek RA 1-N-glycyl βoligosaccharide derivatives as stable intermediates for the formation of glycoconjugate probes. Biochemistry 1992; 31: 10724—10732.

19.

Lindberg AA Vaccination against enteric pathogens: from science to vaccine trials. Curr Opin Microbiol 1998; 1: 116—124. 20. Mäkela PH Vaccines, coming of age after 200 years. FEMS Microbiol Rev 2000; 24: 9—20.

20.

Tan TQ Pneumococcal conjugate vaccines — implications for community antibiotic prescribing. Curr Opin Microbiol 2000; 3: 502—507.

21.

Boutonnier A. , Villeneuve S. , Nato F. , Dassy B. , Fournier JM Preparation, immunogenicity, and protective efficacy, in a murine model, of a conjugate vaccine composed of the polysaccharide moiety of the lipopolysaccharide of Vibrio cholerae O139 bound to tetanus toxoid. Infect Immun 2001; 69: 3488—3493.

22.

Brade L. , Holst O. , Brade H. An artificial glycoconjugate containing the bisphosphorylated glucosamine disaccharide backbone of lipid A binds lipid A monoclonal antibodies. Infect Immun 1993; 61: 4514—4517.

23.

Spevak W. , Dasgupta F. , Hobbs CJ , Nagy JO An efficient synthesis of N-allylglycosylamides from unprotected carbohydrates . J Org Chem 1996; 61: 3417—3422.

24.

Süsskind M. , Brade L. , Brade H. , Holst O. Identification of a novel heptoglycan of alpha1→2-linked D-glycero-D-manno-heptopyranose. Chemical and antigenic structure of lipopolysaccharides from Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-:K20-). J Biol Chem 1998; 273: 7006—7017.

25.

Meyer B. , Weimar T. , Peters T. Screening mixtures for biological activity by NMR. Eur J Biochem 1997; 246: 705—709.