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. 1997 Mar;65(3):916–923. doi: 10.1128/iai.65.3.916-923.1997

Identification of the N-acetylneuraminyllactose-specific laminin-binding protein of Helicobacter pylori.

K H Valkonen 1, T Wadström 1, A P Moran 1
PMCID: PMC175069  PMID: 9038297

Abstract

The interaction of the gastroduodenal pathogen Helicobacter pylori with the glycoprotein laminin was investigated. Binding of 125I-radiolabelled laminin in a liquid-phase assay by both hemagglutinating and poorly hemagglutinating strains was rapid, saturable, specific, partially reversible, of high affinity, and insensitive to pH. Inhibition of laminin binding by fetuin, but not asialofetuin, and reduced bacterial binding to periodate- or sialidase-treated laminin indicated that glycosylation, particularly sialylation, was important for laminin binding by H. pylori. Inhibition experiments with monosaccharides, disaccharides, and trisaccharides showed that the strains bound to a region spanning a trisaccharide. In particular, inhibition and displacement studies showed that binding to the trisaccharide N-acetylneuraminyl-alpha(2-3)-lactose [NeuAc(2-3)Lac] was preferential to that to the NeuAc(2-6)Lac isomer. Complete inhibition of laminin binding by both hemagglutinating and poorly hemagglutinating strains was achieved only when isolated lipopolysaccharide (LPS) was used as an inhibitor in combination with heat or protease treatment of H. pylori cells, thereby confirming the involvement of both LPS and a protein adhesin in laminin binding. Further inhibition experiments indicated that the protein receptor, rather than LPS, on H. pylori bound NeuAc(2-3)Lac. By using a Western blotting procedure, a 25-kDa outer membrane protein was identified as mediating laminin binding by both hemagglutinating and poorly hemagglutinating H. pylori strains. The specificity of binding was confirmed by complete inhibition of laminin binding by the 25-kDa protein with NeuAc(2-3)Lac. The data collectively suggest that a 25-kDa outer membrane protein acts in a lectin-like manner with LPS to mediate attachment of H. pylori to laminin.

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Selected References

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  1. Arumugham R. G., Hsieh T. C., Tanzer M. L., Laine R. A. Structures of the asparagine-linked sugar chains of laminin. Biochim Biophys Acta. 1986 Aug 6;883(1):112–126. doi: 10.1016/0304-4165(86)90142-x. [DOI] [PubMed] [Google Scholar]
  2. Beck K., Hunter I., Engel J. Structure and function of laminin: anatomy of a multidomain glycoprotein. FASEB J. 1990 Feb 1;4(2):148–160. doi: 10.1096/fasebj.4.2.2404817. [DOI] [PubMed] [Google Scholar]
  3. Blaser M. J., Parsonnet J. Parasitism by the "slow" bacterium Helicobacter pylori leads to altered gastric homeostasis and neoplasia. J Clin Invest. 1994 Jul;94(1):4–8. doi: 10.1172/JCI117336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clyne M., Drumm B. Adherence of Helicobacter pylori to primary human gastrointestinal cells. Infect Immun. 1993 Oct;61(10):4051–4057. doi: 10.1128/iai.61.10.4051-4057.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dean J. W., 3rd, Chandrasekaran S., Tanzer M. L. Lectins inhibit cell binding and spreading on a laminin substrate. Biochem Biophys Res Commun. 1988 Oct 14;156(1):411–416. doi: 10.1016/s0006-291x(88)80856-8. [DOI] [PubMed] [Google Scholar]
  6. Dennis J. W., Waller C. A., Schirrmacher V. Identification of asparagine-linked oligosaccharides involved in tumor cell adhesion to laminin and type IV collagen. J Cell Biol. 1984 Oct;99(4 Pt 1):1416–1423. doi: 10.1083/jcb.99.4.1416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Doig P., Austin J. W., Kostrzynska M., Trust T. J. Production of a conserved adhesin by the human gastroduodenal pathogen Helicobacter pylori. J Bacteriol. 1992 Apr;174(8):2539–2547. doi: 10.1128/jb.174.8.2539-2547.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doig P., Austin J. W., Trust T. J. The Helicobacter pylori 19.6-kilodalton protein is an iron-containing protein resembling ferritin. J Bacteriol. 1993 Jan;175(2):557–560. doi: 10.1128/jb.175.2.557-560.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doig P., Trust T. J. Identification of surface-exposed outer membrane antigens of Helicobacter pylori. Infect Immun. 1994 Oct;62(10):4526–4533. doi: 10.1128/iai.62.10.4526-4533.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Emödy L., Carlsson A., Ljungh A., Wadström T. Mannose-resistant haemagglutination by Campylobacter pylori. Scand J Infect Dis. 1988;20(3):353–354. doi: 10.3109/00365548809032466. [DOI] [PubMed] [Google Scholar]
  11. Evans D. G., Evans D. J., Jr, Graham D. Y. Receptor-mediated adherence of Campylobacter pylori to mouse Y-1 adrenal cell monolayers. Infect Immun. 1989 Aug;57(8):2272–2278. doi: 10.1128/iai.57.8.2272-2278.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Evans D. G., Evans D. J., Jr, Moulds J. J., Graham D. Y. N-acetylneuraminyllactose-binding fibrillar hemagglutinin of Campylobacter pylori: a putative colonization factor antigen. Infect Immun. 1988 Nov;56(11):2896–2906. doi: 10.1128/iai.56.11.2896-2906.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Evans D. G., Karjalainen T. K., Evans D. J., Jr, Graham D. Y., Lee C. H. Cloning, nucleotide sequence, and expression of a gene encoding an adhesin subunit protein of Helicobacter pylori. J Bacteriol. 1993 Feb;175(3):674–683. doi: 10.1128/jb.175.3.674-683.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fitzgerald T. J., Repesh L. A., Blanco D. R., Miller J. N. Attachment of Treponema pallidum to fibronectin, laminin, collagen IV, and collagen I, and blockage of attachment by immune rabbit IgG. Br J Vener Dis. 1984 Dec;60(6):357–363. doi: 10.1136/sti.60.6.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Forman D., Newell D. G., Fullerton F., Yarnell J. W., Stacey A. R., Wald N., Sitas F. Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation. BMJ. 1991 Jun 1;302(6788):1302–1305. doi: 10.1136/bmj.302.6788.1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fujiwara S., Shinkai H., Deutzmann R., Paulsson M., Timpl R. Structure and distribution of N-linked oligosaccharide chains on various domains of mouse tumour laminin. Biochem J. 1988 Jun 1;252(2):453–461. doi: 10.1042/bj2520453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Furtado G. C., Slowik M., Kleinman H. K., Joiner K. A. Laminin enhances binding of Toxoplasma gondii tachyzoites to J774 murine macrophage cells. Infect Immun. 1992 Jun;60(6):2337–2342. doi: 10.1128/iai.60.6.2337-2342.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gehlsen K. R., Dillner L., Engvall E., Ruoslahti E. The human laminin receptor is a member of the integrin family of cell adhesion receptors. Science. 1988 Sep 2;241(4870):1228–1229. doi: 10.1126/science.2970671. [DOI] [PubMed] [Google Scholar]
  19. Graham D. Y., Lew G. M., Klein P. D., Evans D. G., Evans D. J., Jr, Saeed Z. A., Malaty H. M. Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. A randomized, controlled study. Ann Intern Med. 1992 May 1;116(9):705–708. doi: 10.7326/0003-4819-116-9-705. [DOI] [PubMed] [Google Scholar]
  20. Hazell S. L., Lee A., Brady L., Hennessy W. 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):658–663. doi: 10.1093/infdis/153.4.658. [DOI] [PubMed] [Google Scholar]
  21. Hessey S. J., Spencer J., Wyatt J. I., Sobala G., Rathbone B. J., Axon A. T., Dixon M. F. Bacterial adhesion and disease activity in Helicobacter associated chronic gastritis. Gut. 1990 Feb;31(2):134–138. doi: 10.1136/gut.31.2.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huang J., Keeling P. W., Smyth C. J. Identification of erythrocyte-binding antigens in Helicobacter pylori. J Gen Microbiol. 1992 Jul;138(7):1503–1513. doi: 10.1099/00221287-138-7-1503. [DOI] [PubMed] [Google Scholar]
  23. Karch H., Leying H., Büscher K. H., Kroll H. P., Opferkuch W. Isolation and separation of physicochemically distinct fimbrial types expressed on a single culture of Escherichia coli O7:K1:H6. Infect Immun. 1985 Feb;47(2):549–554. doi: 10.1128/iai.47.2.549-554.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Knibbs R. N., Perini F., Goldstein I. J. Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin. Biochemistry. 1989 Jul 25;28(15):6379–6392. doi: 10.1021/bi00441a034. [DOI] [PubMed] [Google Scholar]
  25. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Lee A., Fox J., Hazell S. Pathogenicity of Helicobacter pylori: a perspective. Infect Immun. 1993 May;61(5):1601–1610. doi: 10.1128/iai.61.5.1601-1610.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liotta L. A., Rao C. N., Wewer U. M. Biochemical interactions of tumor cells with the basement membrane. Annu Rev Biochem. 1986;55:1037–1057. doi: 10.1146/annurev.bi.55.070186.005133. [DOI] [PubMed] [Google Scholar]
  29. Liukkonen J., Haataja S., Tikkanen K., Kelm S., Finne J. Identification of N-acetylneuraminyl alpha 2-->3 poly-N-acetyllactosamine glycans as the receptors of sialic acid-binding Streptococcus suis strains. J Biol Chem. 1992 Oct 15;267(29):21105–21111. [PubMed] [Google Scholar]
  30. Lopes J. D., dos Reis M., Brentani R. R. Presence of laminin receptors in Staphylococcus aureus. Science. 1985 Jul 19;229(4710):275–277. doi: 10.1126/science.3160113. [DOI] [PubMed] [Google Scholar]
  31. Markwell M. A. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labeling of antiserum. Anal Biochem. 1982 Sep 15;125(2):427–432. doi: 10.1016/0003-2697(82)90025-2. [DOI] [PubMed] [Google Scholar]
  32. Marre R., Hacker J. Role of S- and common-type I-fimbriae of Escherichia coli in experimental upper and lower urinary tract infection. Microb Pathog. 1987 Mar;2(3):223–226. doi: 10.1016/0882-4010(87)90023-4. [DOI] [PubMed] [Google Scholar]
  33. Marshall B. J., Warren J. R. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984 Jun 16;1(8390):1311–1315. doi: 10.1016/s0140-6736(84)91816-6. [DOI] [PubMed] [Google Scholar]
  34. Moran A. P. Cell surface characteristics of Helicobacter pylori. FEMS Immunol Med Microbiol. 1995 Feb;10(3-4):271–280. doi: 10.1111/j.1574-695X.1995.tb00043.x. [DOI] [PubMed] [Google Scholar]
  35. Moran A. P., Helander I. M., Kosunen T. U. Compositional analysis of Helicobacter pylori rough-form lipopolysaccharides. J Bacteriol. 1992 Feb;174(4):1370–1377. doi: 10.1128/jb.174.4.1370-1377.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nomura A., Stemmermann G. N., Chyou P. H., Kato I., Perez-Perez G. I., Blaser M. J. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med. 1991 Oct 17;325(16):1132–1136. doi: 10.1056/NEJM199110173251604. [DOI] [PubMed] [Google Scholar]
  37. O'Toole P. W., Janzon L., Doig P., Huang J., Kostrzynska M., Trust T. J. The putative neuraminyllactose-binding hemagglutinin HpaA of Helicobacter pylori CCUG 17874 is a lipoprotein. J Bacteriol. 1995 Nov;177(21):6049–6057. doi: 10.1128/jb.177.21.6049-6057.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  39. Parkkinen J., Rogers G. N., Korhonen T., Dahr W., Finne J. Identification of the O-linked sialyloligosaccharides of glycophorin A as the erythrocyte receptors for S-fimbriated Escherichia coli. Infect Immun. 1986 Oct;54(1):37–42. doi: 10.1128/iai.54.1.37-42.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Parsonnet J., Hansen S., Rodriguez L., Gelb A. B., Warnke R. A., Jellum E., Orentreich N., Vogelman J. H., Friedman G. D. Helicobacter pylori infection and gastric lymphoma. N Engl J Med. 1994 May 5;330(18):1267–1271. doi: 10.1056/NEJM199405053301803. [DOI] [PubMed] [Google Scholar]
  41. Pikkarainen T., Schulthess T., Engel J., Tryggvason K. Recombinant laminin B1 chains exhibit intact short-arm domains but do not form oligomeric molecules. Eur J Biochem. 1992 Oct 15;209(2):571–582. doi: 10.1111/j.1432-1033.1992.tb17322.x. [DOI] [PubMed] [Google Scholar]
  42. Plotkowski M. C., Tournier J. M., Puchelle E. Pseudomonas aeruginosa strains possess specific adhesins for laminin. Infect Immun. 1996 Feb;64(2):600–605. doi: 10.1128/iai.64.2.600-605.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rauws E. A., Tytgat G. N. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet. 1990 May 26;335(8700):1233–1235. doi: 10.1016/0140-6736(90)91301-p. [DOI] [PubMed] [Google Scholar]
  44. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  45. Sankaran K., Ramachandran V., Subrahmanyam Y. V., Rajarathnam S., Elango S., Roy R. K. Congo red-mediated regulation of levels of Shigella flexneri 2a membrane proteins. Infect Immun. 1989 Aug;57(8):2364–2371. doi: 10.1128/iai.57.8.2364-2371.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Slomiany B. L., Piotrowski J., Rajiah G., Slomiany A. Inhibition of gastric mucosal laminin receptor by Helicobacter pylori lipopolysaccharide: effect of nitecapone. Gen Pharmacol. 1991;22(6):1063–1069. doi: 10.1016/0306-3623(91)90578-t. [DOI] [PubMed] [Google Scholar]
  47. Smit H., Gaastra W., Kamerling J. P., Vliegenthart J. F., de Graaf F. K. Isolation and structural characterization of the equine erythrocyte receptor for enterotoxigenic Escherichia coli K99 fimbrial adhesin. Infect Immun. 1984 Nov;46(2):578–584. doi: 10.1128/iai.46.2.578-584.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sommer P., Gleyzal C., Guerret S., Etienne J., Grimaud J. A. Induction of a putative laminin-binding protein of Streptococcus gordonii in human infective endocarditis. Infect Immun. 1992 Feb;60(2):360–365. doi: 10.1128/iai.60.2.360-365.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Steer H. W. The gastro-duodenal epithelium in peptic ulceration. J Pathol. 1985 Aug;146(4):355–362. doi: 10.1002/path.1711460409. [DOI] [PubMed] [Google Scholar]
  50. Switalski L. M., Murchison H., Timpl R., Curtiss R., 3rd, Hök M. Binding of laminin to oral and endocarditis strains of viridans streptococci. J Bacteriol. 1987 Mar;169(3):1095–1101. doi: 10.1128/jb.169.3.1095-1101.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Switalski L. M., Speziale P., Hök M., Wadström T., Timpl R. Binding of Streptococcus pyogenes to laminin. J Biol Chem. 1984 Mar 25;259(6):3734–3738. [PubMed] [Google Scholar]
  52. Taylor D. N., Blaser M. J. The epidemiology of Helicobacter pylori infection. Epidemiol Rev. 1991;13:42–59. doi: 10.1093/oxfordjournals.epirev.a036078. [DOI] [PubMed] [Google Scholar]
  53. Timpl R. Structure and biological activity of basement membrane proteins. Eur J Biochem. 1989 Apr 1;180(3):487–502. doi: 10.1111/j.1432-1033.1989.tb14673.x. [DOI] [PubMed] [Google Scholar]
  54. Trust T. J., Doig P., Emödy L., Kienle Z., Wadström T., O'Toole P. High-affinity binding of the basement membrane proteins collagen type IV and laminin to the gastric pathogen Helicobacter pylori. Infect Immun. 1991 Dec;59(12):4398–4404. doi: 10.1128/iai.59.12.4398-4404.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983 Jun 4;1(8336):1273–1275. [PubMed] [Google Scholar]
  56. Valkonen K. H., Ringner M., Ljungh A., Wadström T. High-affinity binding of laminin by Helicobacter pylori: evidence for a lectin-like interaction. FEMS Immunol Med Microbiol. 1993 Jun;7(1):29–37. doi: 10.1111/j.1574-695X.1993.tb00378.x. [DOI] [PubMed] [Google Scholar]
  57. Valkonen K. H., Veijola J., Dagberg B., Uhlin B. E. Binding of basement-membrane laminin by Escherichia coli. Mol Microbiol. 1991 Sep;5(9):2133–2141. doi: 10.1111/j.1365-2958.1991.tb02143.x. [DOI] [PubMed] [Google Scholar]
  58. Valkonen K. H., Wadström T., Moran A. P. Interaction of lipopolysaccharides of Helicobacter pylori with basement membrane protein laminin. Infect Immun. 1994 Sep;62(9):3640–3648. doi: 10.1128/iai.62.9.3640-3648.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vuento M., Vaheri A. Purification of fibronectin from human plasma by affinity chromatography under non-denaturing conditions. Biochem J. 1979 Nov 1;183(2):331–337. doi: 10.1042/bj1830331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Westerlund B., Merenmies J., Rauvala H., Miettinen A., Järvinen A. K., Virkola R., Holthöfer H., Korhonen T. K. The O75X adhesin of uropathogenic Escherichia coli: receptor-active domains in the canine urinary tract and in-vitro interaction with laminin. Microb Pathog. 1987 Aug;3(2):117–127. doi: 10.1016/0882-4010(87)90070-2. [DOI] [PubMed] [Google Scholar]
  61. Wotherspoon A. C., Ortiz-Hidalgo C., Falzon M. R., Isaacson P. G. Helicobacter pylori-associated gastritis and primary B-cell gastric lymphoma. Lancet. 1991 Nov 9;338(8776):1175–1176. doi: 10.1016/0140-6736(91)92035-z. [DOI] [PubMed] [Google Scholar]
  62. Yurchenco P. D., Schittny J. C. Molecular architecture of basement membranes. FASEB J. 1990 Apr 1;4(6):1577–1590. doi: 10.1096/fasebj.4.6.2180767. [DOI] [PubMed] [Google Scholar]
  63. Zhou Q., Cummings R. D. The S-type lectin from calf heart tissue binds selectively to the carbohydrate chains of laminin. Arch Biochem Biophys. 1990 Aug 15;281(1):27–35. doi: 10.1016/0003-9861(90)90408-q. [DOI] [PubMed] [Google Scholar]

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