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. 1983 Feb;153(2):955–961. doi: 10.1128/jb.153.2.955-961.1983

Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein.

E A Worobec, A K Taneja, R S Hodges, W Paranchych
PMCID: PMC221719  PMID: 6185467

Abstract

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.

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Selected References

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  1. Armstrong G. D., Frost L. S., Sastry P. A., Paranchych W. Comparative biochemical studies on F and EDP208 conjugative pili. J Bacteriol. 1980 Jan;141(1):333–341. doi: 10.1128/jb.141.1.333-341.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong G. D., Frost L. S., Vogel H. J., Paranchych W. Nature of the carbohydrate and phosphate associated with ColB2 and EDP208 pilin. J Bacteriol. 1981 Mar;145(3):1167–1176. doi: 10.1128/jb.145.3.1167-1176.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Atassi M. Z. Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigenic structures of proteins. Immunochemistry. 1975 May;12(5):423–438. doi: 10.1016/0019-2791(75)90010-5. [DOI] [PubMed] [Google Scholar]
  4. Chong P. C., Hodges R. S. A new heterobifunctional cross-linking reagent for the study of biological interactions between proteins. I. Design, synthesis, and characterization. J Biol Chem. 1981 May 25;256(10):5064–5070. [PubMed] [Google Scholar]
  5. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  6. Folkhard W., Leonard K. R., Malsey S., Marvin D. A., Dubochet J., Engel A., Achtman M., Helmuth R. X-ray diffraction and electron microscope studies on the structure of bacterial F pili. J Mol Biol. 1979 May 15;130(2):145–160. doi: 10.1016/0022-2836(79)90423-6. [DOI] [PubMed] [Google Scholar]
  7. Frost L. S., Armstrong G. D., Finlay B. B., Edwards B. F., Paranchych W. N-terminal amino acid sequencing of EDP208 conjugative pili. J Bacteriol. 1983 Feb;153(2):950–954. doi: 10.1128/jb.153.2.950-954.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gutte B., Merrifield R. B. The synthesis of ribonuclease A. J Biol Chem. 1971 Mar 25;246(6):1922–1941. [PubMed] [Google Scholar]
  9. Hodges R. S., Merrifield R. B. Monitoring of solid phase peptide synthesis by an automated spectrophotometric picrate method. Anal Biochem. 1975 May 12;65(1-2):241–272. doi: 10.1016/0003-2697(75)90509-6. [DOI] [PubMed] [Google Scholar]
  10. Hodges R. S., Merrifield R. B. The role of serine-123 in the activity and specificity of ribonuclease. Reactivation of ribonuclease 1-118 by the synthetic COOH-terminal tetradecapeptide, ribonuclease 111-124, and its O-methylserine and alanine analogs. J Biol Chem. 1975 Feb 25;250(4):1231–1241. [PubMed] [Google Scholar]
  11. Hodges R. S., Saund A. K., Chong P. C., St-Pierre S. A., Reid R. E. Synthetic model for two-stranded alpha-helical coiled-coils. Design, synthesis, and characterization of an 86-residue analog of tropomyosin. J Biol Chem. 1981 Feb 10;256(3):1214–1224. [PubMed] [Google Scholar]
  12. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klemm P. The complete amino-acid sequence of the K88 antigen, a fimbrial protein from Escherichia coli. Eur J Biochem. 1981 Jul;117(3):617–627. doi: 10.1111/j.1432-1033.1981.tb06382.x. [DOI] [PubMed] [Google Scholar]
  14. Nagy B., Moon H. W., Isaacson R. E., To C. C., Brinton C. C. Immunization of suckling pigs against enteric enterotoxigenic Escherichia coli infection by vaccinating dams with purified pili. Infect Immun. 1978 Jul;21(1):269–274. doi: 10.1128/iai.21.1.269-274.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Poljak R. J., Amzel L. M., Chen B. L., Chiu Y. Y., Phizackerley R. P., Saul F., Ysern X. Three-dimensional structure and diversity of immunoglobulins. Cold Spring Harb Symp Quant Biol. 1977;41(Pt 2):639–645. doi: 10.1101/sqb.1977.041.01.073. [DOI] [PubMed] [Google Scholar]
  16. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Voller A., Bidwell D., Huldt G., Engvall E. A microplate method of enzyme-linked immunosorbent assay and its application to malaria. Bull World Health Organ. 1974;51(2):209–211. [PMC free article] [PubMed] [Google Scholar]

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