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PMID: 7928954 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori.

Journal of bacteriology ·Vol. 176 ·No. 19 ·1994-10-00 ·Pages 5938-48

Kostrzynska M, O'Toole PW, Taylor DE, Trust TJ

Abstract

Antisera raised in rabbits to whole cells of Helicobacter pylori recognized as a major antigen a protein with an apparent molecular weight of 20,000. The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate followed by molecular sieving. The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry. The lpp20 gene encoding this protein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the native H. pylori promoter. lpp20 was mapped to the same 358-kb NruI fragment as flaB. DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,094 containing a 21-residue typical lipoprotein signal peptide and consensus prolipoprotein processing site. The mass of the deduced 154-residue mature protein was 16,865 Da. Growth of E. coli cells expressing the cloned H. pylori lpp20 gene in the presence of [3H]palmitic acid resulted in radiolabelled Lpp20 while treatment of the E. coli cells with globomycin caused accumulation of unprocessed Lpp20, consistent with Lpp20 being a lipoprotein. Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also found in the outer membrane. A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonessential class of lipoproteins.

Related Genes
lpp20
MeSH Terms
Amino Acid Sequence Antigens, Bacterial/genetics,isolation & purification Bacterial Proteins/genetics Base Sequence Cell Compartmentation Cloning, Molecular Conserved Sequence Helicobacter pylori/genetics,immunology Lipoproteins/genetics,isolation & purification Membrane Proteins/genetics Molecular Sequence Data Mutagenesis, Insertional Protein Conformation Protein Processing, Post-Translational Restriction Mapping Sequence Analysis, DNA Sequence Homology, Amino Acid
Chemicals
Antigens, Bacterial Bacterial Proteins Lipoproteins Lpp20 protein, Helicobacter pylori Membrane Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Kostrzynska M
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
O'Toole P W
Taylor D E
Trust T J
References (76)
76 references, click to expand
  1. Campylobacter pyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium.
    J Infect Dis. 1986 Apr;153(4):658-63 PMID: 3950447
  2. Structural requirement at the cleavage site for efficient processing of the lipoprotein secretory precursor of Escherichia coli.
    J Biol Chem. 1986 Aug 25;261(24):10970-5 PMID: 3525558
  3. Biogenesis of lipoproteins in bacteria.
    Curr Top Microbiol Immunol. 1986;125:127-57 PMID: 3527576
  4. GC content of DNA of Campylobacter pylori and other species belonging or related to the genus Campylobacter.
    Ann Inst Pasteur Microbiol. 1988 Sep-Oct;139(5):527-34 PMID: 3252903
  5. Comparison of methods used to separate the inner and outer membranes of cell envelopes of Campylobacter spp.
    J Gen Microbiol. 1988 Nov;134(11):2925-32 PMID: 2474628
  6. Natural competence for genetic transformation in Campylobacter pylori.
    J Infect Dis. 1990 Feb;161(2):365-6 PMID: 2299221
  7. Direct protein microsequencing from Immobilon-P Transfer Membrane.
    Biotechniques. 1988 Feb;6(2):154-9 PMID: 3273181
  8. Helicobacter pylori and the pathogenesis of gastroduodenal inflammation.
    J Infect Dis. 1990 Apr;161(4):626-33 PMID: 2181029
  9. Purification and characterization of urease from Helicobacter pylori.
    J Biol Chem. 1990 Jun 5;265(16):9464-9 PMID: 2188975
  10. Lipoproteins in bacteria.
    J Bioenerg Biomembr. 1990 Jun;22(3):451-71 PMID: 2202727
  11. Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580.
    J Bacteriol. 1990 Sep;172(9):5211-7 PMID: 2168376
  12. Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction.
    Gene. 1990 Sep 28;94(1):23-8 PMID: 2227449
  13. Paracrystalline inclusions of a novel ferritin containing nonheme iron, produced by the human gastric pathogen Helicobacter pylori: evidence for a third class of ferritins.
    J Bacteriol. 1993 Feb;175(4):966-72 PMID: 8432720
  14. 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):4350-4 PMID: 388439
  15. Nucleotide sequence of the gene for the peptidoglycan-associated lipoprotein of Escherichia coli K12.
    Eur J Biochem. 1987 Feb 16;163(1):73-7 PMID: 3545827
  16. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications.
    Nucleic Acids Res. 1987 Feb 11;15(3):1281-95 PMID: 3547335
  17. New and versatile cloning vectors with kanamycin-resistance marker.
    Gene. 1987;56(2-3):309-12 PMID: 3315864
  18. Biologic activities of antibody to a peptidoglycan-associated lipoprotein of Haemophilus influenzae against multiple clinical isolates of H. influenzae type b.
    Infect Immun. 1987 Dec;55(12):2878-83 PMID: 3316025
  19. Cloning and sequencing of Haemophilus influenzae outer membrane protein P6.
    Infect Immun. 1988 Jan;56(1):128-34 PMID: 3257200
  20. A single amino acid determinant of the membrane localization of lipoproteins in E. coli.
    Cell. 1988 May 6;53(3):423-32 PMID: 3284654
  21. Expression of the gene encoding the 17-kilodalton antigen from Rickettsia rickettsii: transcription and posttranslational modification.
    J Bacteriol. 1988 Oct;170(10):4493-500 PMID: 3139629
  22. Predicting the secondary structure of globular proteins using neural network models.
    J Mol Biol. 1988 Aug 20;202(4):865-84 PMID: 3172241
  23. Protein secondary structure prediction with a neural network.
    Proc Natl Acad Sci U S A. 1989 Jan;86(1):152-6 PMID: 2911565
  24. Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins.
    Infect Immun. 1989 Jun;57(6):1825-33 PMID: 2722241
  25. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  26. Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes.
    J Immunol. 1972 Jul;109(1):129-35 PMID: 4113792
  27. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate.
    J Bacteriol. 1973 Sep;115(3):717-22 PMID: 4580564
  28. The nicotinic cholinergic receptor : different compositions evidenced by statistical analysis.
    Biochem Biophys Res Commun. 1975 Jan 20;62(2):407-14 PMID: 1111530
  29. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis.
    J Biol Chem. 1977 Feb 10;252(3):1102-6 PMID: 320200
  30. Mechanism of action of globomycin.
    J Antibiot (Tokyo). 1978 Nov;31(11):1203-5 PMID: 721715
  31. Prediction of the secondary structure of proteins from their amino acid sequence.
    Adv Enzymol Relat Areas Mol Biol. 1978;47:45-148 PMID: 364941
  32. Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production.
    Infect Immun. 1993 May;61(5):1799-809 PMID: 8478069
  33. Cloning and genetic characterization of the Helicobacter pylori and Helicobacter mustelae flaB flagellin genes and construction of H. pylori flaA- and flaB-negative mutants by electroporation-mediated allelic exchange.
    J Bacteriol. 1993 Jun;175(11):3278-88 PMID: 8501031
  34. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer.
    Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5791-5 PMID: 8516329
  35. Construction of new Campylobacter cloning vectors and a new mutational cat cassette.
    Gene. 1993 Aug 16;130(1):127-30 PMID: 8344519
  36. Prediction of protein secondary structure at better than 70% accuracy.
    J Mol Biol. 1993 Jul 20;232(2):584-99 PMID: 8345525
  37. Borrelia burgdorferi outer surface lipoproteins OspA and OspB possess B-cell mitogenic and cytokine-stimulatory properties.
    Infect Immun. 1993 Sep;61(9):3843-53 PMID: 8359905
  38. Molecular and immunological characterization of a novel polymorphic lipoprotein of Borrelia burgdorferi.
    Infect Immun. 1993 Oct;61(10):4158-66 PMID: 8104894
  39. Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae.
    Infect Immun. 1991 Feb;59(2):600-8 PMID: 1987077
  40. Isolation and biochemical and molecular analyses of a species-specific protein antigen from the gastric pathogen Helicobacter pylori.
    J Bacteriol. 1991 Jan;173(2):505-13 PMID: 1987145
  41. Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.
    J Bacteriol. 1991 Feb;173(3):937-46 PMID: 1704004
  42. Soluble surface proteins from Helicobacter pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism.
    J Clin Invest. 1991 Mar;87(3):894-900 PMID: 1847939
  43. Membrane topology of Escherichia coli prolipoprotein signal peptidase (signal peptidase II).
    J Biol Chem. 1991 Sep 15;266(26):17667-72 PMID: 1894646
  44. Gastritis induced by Helicobacter pylori in gnotobiotic piglets.
    Rev Infect Dis. 1991 Jul-Aug;13 Suppl 8:S681-5 PMID: 1925308
  45. Characterization of an immunoreactive species-specific 19-kilodalton outer membrane protein from Helicobacter pylori by using a monoclonal antibody.
    J Clin Microbiol. 1991 Aug;29(8):1620-4 PMID: 1761682
  46. Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation.
    Gastroenterology. 1992 Feb;102(2):720-7 PMID: 1732141
  47. Molecular cloning, nucleotide sequence, and characterization of a 40,000-molecular-weight lipoprotein of Haemophilus somnus.
    Infect Immun. 1992 Mar;60(3):826-31 PMID: 1541556
  48. Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum.
    Infect Immun. 1992 Apr;60(4):1568-76 PMID: 1372297
  49. Structural determinants in addition to the amino-terminal sorting sequence influence membrane localization of Escherichia coli lipoproteins.
    J Bacteriol. 1992 Apr;174(7):2095-101 PMID: 1551832
  50. Identification and purification of a cpn60 heat shock protein homolog from Helicobacter pylori.
    Infect Immun. 1992 May;60(5):1946-51 PMID: 1563786
  51. Purification and characterization of the vacuolating toxin from Helicobacter pylori.
    J Biol Chem. 1992 May 25;267(15):10570-5 PMID: 1587837
  52. Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level.
    J Bacteriol. 1992 Nov;174(21):6800-6 PMID: 1400229
  53. Structural comparison of urease and a GroEL analog from Helicobacter pylori.
    J Bacteriol. 1992 Nov;174(22):7470-3 PMID: 1358875
  54. Synthetic lipopeptides as novel adjuvants.
    Res Immunol. 1992 Jun;143(5):548-53; discussion 579-80 PMID: 1439136
  55. Solid phase peptide synthesis of lipopeptide vaccines eliciting epitope-specific B-, T-helper and T-killer cell response.
    Int J Pept Protein Res. 1992 Sep-Oct;40(3-4):255-60 PMID: 1282504
  56. Cloning, nucleotide sequence, and expression of a gene encoding an adhesin subunit protein of Helicobacter pylori.
    J Bacteriol. 1993 Feb;175(3):674-83 PMID: 7678592
  57. Nucleotide sequence analysis and serologic characterization of a 27-kilodalton Mycobacterium intracellulare lipoprotein.
    Infect Immun. 1993 Mar;61(3):1074-81 PMID: 8432589
  58. Helicobacter pylori: microbiology of a 'slow' bacterial infection.
    Trends Microbiol. 1993 Oct;1(7):255-60 PMID: 8162405
  59. Identification and molecular characterization of a major ring-forming surface protein from the gastric pathogen Helicobacter mustelae.
    Mol Microbiol. 1994 Jan;11(2):349-61 PMID: 8170397
  60. Identification of surface-exposed outer membrane antigens of Helicobacter pylori.
    Infect Immun. 1994 Oct;62(10):4526-33 PMID: 7927718
  61. The role of the lipoprotein sorting signal (aspartate +2) in pullulanase secretion.
    Mol Microbiol. 1993 Sep;9(5):1061-9 PMID: 7934912
  62. Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases.
    J Biol Chem. 1980 May 10;255(9):3964-76 PMID: 6768745
  63. A simple method for displaying the hydropathic character of a protein.
    J Mol Biol. 1982 May 5;157(1):105-32 PMID: 7108955
  64. Structural prediction of membrane-bound proteins.
    Eur J Biochem. 1982 Nov 15;128(2-3):565-75 PMID: 7151796
  65. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.
    Gene. 1982 Oct;19(3):259-68 PMID: 6295879
  66. Unidentified curved bacilli on gastric epithelium in active chronic gastritis.
    Lancet. 1983 Jun 4;1(8336):1273-5 PMID: 6134060
  67. Molecular identification of surface protein antigens of Campylobacter jejuni.
    Infect Immun. 1983 Nov;42(2):675-82 PMID: 6642648
  68. Lambda replacement vectors carrying polylinker sequences.
    J Mol Biol. 1983 Nov 15;170(4):827-42 PMID: 6315951
  69. A distinct signal peptidase for prolipoprotein in Escherichia coli.
    J Cell Biochem. 1984;24(2):113-20 PMID: 6373799
  70. Prolipoprotein modification and processing in Escherichia coli. A unique secondary structure in prolipoprotein signal sequence for the recognition by glyceryl transferase.
    Eur J Biochem. 1984 Jun 1;141(2):331-7 PMID: 6428886
  71. Three-dimensional structure of membrane and surface proteins.
    Annu Rev Biochem. 1984;53:595-623 PMID: 6383201
  72. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
    Gene. 1985;33(1):103-19 PMID: 2985470
  73. The detection and classification of membrane-spanning proteins.
    Biochim Biophys Acta. 1985 May 28;815(3):468-76 PMID: 3838905
  74. Inhibition of prolipoprotein signal peptidase by globomycin.
    J Biol Chem. 1985 May 25;260(10):5891-4 PMID: 3888977
  75. Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope.
    J Biol Chem. 1986 Jan 5;261(1):428-43 PMID: 3510202
  76. Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli. Cloning, DNA sequence, and expression of its gene.
    J Biol Chem. 1986 Feb 15;261(5):2284-8 PMID: 3003106
Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.176.19.5938-5948.1994
Published
1994-10-00
Pages
5938-48
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC196810
Subset
IM
Grants
PHS HHS · R01A129927-01A2 · United States
Databases
GENBANK
U12429
External Links
PubMed source DOI: 10.1128/jb.176.19.5938-5948.1994 PMC Full Text View journal details
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